Granulysin in immunotherapy

ABSTRACT

Methods of stimulating or enhancing an immune response in a host are disclosed. The methods include contacting a monocyte with 15 kD granulysin thereby producing a monocyte-derived dendritic cell. In one example, the method further includes contacting the monocyte or monocyte-derived dendritic cell with a target antigen, such as a tumor antigen or an autoimmune antigen. In another embodiment, the method includes contacting the monocyte with an additional agent that enhances maturation of dendritic cells or induces immunological tolerance. The methods are of use in vivo, in vitro and ex vivo. In another aspect, the disclosure relates to compositions and methods for the treatment of tumors.

CROSS REFERENCE TO RELATED APPLICATION

This is a divisional of U.S. application Ser. No. 14/341,317, filed Jul.25, 2014, which is a divisional of U.S. application Ser. No. 13/501,726,filed Apr. 12, 2012, now U.S. Pat. No. 8,815,229, issued Aug. 26, 2014,which is the §371 U.S. National Stage of International Application No.PCT/US2010/052036, filed Oct. 8, 2010, which was published in Englishunder PCT Article 21(2), which in turn claims the benefit of U.S.Provisional Application No. 61/250,601, filed Oct. 12, 2009, all ofwhich are incorporated by reference herein in their entirety.

FIELD

This disclosure relates to methods for the treatment of immune-baseddisorders, such as autoimmune diseases, organ transplantation rejectionand tumor immunotherapy. The disclosure also relates to the stimulationof an immune response in a host upon administration of a therapeuticallyeffective amount of 15 kD granulysin.

BACKGROUND

Vaccination protocols have improved over the last several decades;however a therapeutically effective immune response has still beendifficult to generate for some conditions. For example, human tumorimmunotherapy has met with only limited success. Among the reasons forthis difficulty have been the limited availability of tumor associatedantigens, and an inability to deliver antigens in a manner that rendersthem immunogenic.

Dendritic cells (DC) include a heterogeneous family of antigenpresenting cells (APC) that present antigens for the modulation of animmune response or induce immunological tolerance. The number ofdendritic cells in the blood is surprisingly few, less than about 1% ofblood mononuclear leukocytes. Thus, the low number of circulatingdendritic cells makes their therapeutic use for the stimulation ormodulation of an immune response difficult. Dendritic precursor cells,such as monocytes, migrate from a host's bone marrow to specific siteswhere they become activated and differentiate into dendritic cells.Following exposure to an antigen and an activation signal, the dendriticcells secrete chemokines and cytokines, and then present the processedantigen to naive T cells to produce an immune response in the host.

Bidirectional interactions between antigen presenting dendritic cellsand naïve T cells initiate either an immunogenic or a tolerance pathwaythat are of particular importance in autoimmune disease and intransplantation medicine. Conventional subsets of dendritic cellsdescribed in humans include myeloid dendritic cells (mDC) andplasmacytoid dendritic cells (pDC).

Dendritic cells possess a distinct morphology and are characterized bythe expression of large amounts of class II MHC antigens, and theabsence of lineage markers, including CD14 (monocyte), CD3 (T cell),CD19, CD20, CD24 (B cells), CD56 (natural killer), and CD66b(granulocyte) (Shortman and Liu, Nat. Rev. Immunol. 2:151-161, 2002).Dendritic cells also express a variety of adhesion and co-stimulatorymolecules such as CD80 and CD86, and molecules that regulateco-stimulation, such as CD40. The phenotype of dendritic cells varieswith the stage of dendritic cell maturation and activation (Chapuis etal., Eur. J. Immunol. 27:431-441, 1997). However, expression of adhesionmolecules, MHC antigens and co-stimulatory molecules increases withdendritic cell maturation. Antibodies that preferentially staindendritic cells are commercially available, such as anti-CD83 andanti-CD80. Accordingly, the expression level of a particular antigenmarker can be used to confirm if the antigen presenting cell is adendritic cell, and if the dendritic cell is mature (Zhou and Tedder, J.Immunol. 154:3821-3835, 1995; Weissman et al., J. Immunol.155:4111-4117, 1995).

Several in vitro methods have been developed to expand populations ofdendritic cells and to augment anti-cancer immunity. Ex vivo exposure ofexpanded populations of dendritic cells to antigens found on tumor cellsor other disease-causing cells, followed by reintroduction of theantigen-loaded dendritic cells to the subject, significantly enhancedpresentation of the antigen to responding T cells. For example,culturing blood mononuclear leukocytes for eight days in the presence ofgranulocyte-monocyte colony stimulating factor (GM-CSF) andinterleukin-4 (IL-4) was found to produce large numbers of dendriticcells (Sallusto and Lanzavecchia, J. Exp. Med. 179:1109-1118, 1994).

DNA vaccines that incorporate plasmids encoding cytokines (such asGM-CSF and IL-4) have been used to investigate dendritic cell maturationpathways. In particular, GM-CSF cDNA has been used as a DNA vaccineadjuvant for glycoprotein B of Pseudorabies virus (PrV) in a murinemouse model (Yoon et al, Microbiol. Immunol. 50:83-92, 2006). At leastnine cytokine-secreting vectors have been identified as geneticadjuvants for DNA vaccines (in “DNA Vaccines Methods and Protocols,”edited by Douglas Lowrie and Robert Whalen).

Nucleic acid immunization is a relatively recent approach in vaccinedevelopment. The ability of DNA vaccines to protect against challengesfrom pathogens has been demonstrated in animal models of influenza,malaria, mycobacterium, HIV, and Ebola. A DNA-based vaccine usuallycomprises purified plasmid DNA carrying sequences encoding a targetantigen under the control of a eukaryotic promoter. The plasmid isinjected into the muscle or skin and the host cells take up the plasmidand express the antigen intracellularly. Expression of the encodedantigen by the host's cells is one of the advantages of this approachbecause it mimics natural infection. To enhance immune responses inducedby DNA vaccines, co-administration of adjuvants such as cytokines,chemokines and co-stimulatory molecules have been used. It is thereforebelieved that administering plasmids encoding cytokines (such as GM-CSFor IL-4) and a target antigen may cause intracellular expression of boththe antigen and the cytokine in the host, thereby providing an enhancedimmune response in the host.

Cancers are a significant public health problem. Many cancer treatmentsare available to such patients, including surgical excision,chemotherapy, radiotherapy, and bone marrow transplantation. While manyconventional cancer therapies are often effective in reducing neoplasticgrowth, healthy cells are frequently compromised by cytotoxictreatments. Non-selective cell damage causes pain, inflammation, hairloss, immunosuppression and gastrointestinal damage. Improvedcompositions and methods are needed to treat, inhibit, or alleviate thedevelopment of tumors. For example, tumor antigens have beenadministered to a tumor bearing host in attempts to produce an immuneresponse to the tumor cells in the host. This approach has met withvarying and modest results.

Granulysin is a naturally occurring protein expressed in human cytotoxicT lymphocytes (CTL) and natural killer (NK) cells. Granulysin expressedin its full-length form has a molecular weight of approximately 15,000Daltons and is known as 15 kD or 15 kDa granulysin. A post-translationalmodified form of 15 kD granulysin in which both the N- and C-termini arecleaved is known as 9 kD granulysin. The 9 kD granulysin peptide hasbeen extensively studied and is observed to possess anti-microbial andtumorcidal activity (Hanson et al., Mol. Immunol. 36:413-422, 1999;Krensky, Biocehm. Pharmacol. 59:317-320, 2000; Clayberger et al., Curr.Opin. Immunol. 15:560-565, 2003; Deng et al., J. Immunol. 174:5243-5248,2005; Stenger et al., Science 282: 121-125, 1998; and Huang et al., J.Immunol. 178:77-84, 2007). The 9 kD granulysin peptide is also known tohave cytolytic properties and its resulting toxicity may limit itstherapeutic use.

SUMMARY

The present disclosure provides a method for stimulating an immuneresponse, or enhancing the efficacy of a vaccine, without simultaneouslyinitiating a cytolytic response in a host. Several publications disclosethat 9 kD granulysin is a cytolytic and antimicrobial compound. However,until recently, the properties of the full-length 15 kD granulysinprotein were unknown. One reason for the lack of research was because ananimal model did not exist (mice do not express granulysin). Inaddition, others reported substantial technical issues when trying toconstitutively express the full-length 15 kD protein in vitro.

Chen et al. (U.S. Patent Publication No. 2008/0050382 A1) identified 15kD granulysin in blister fluids from skin lesions of Stevens-JohnsonSyndrome (SJS) and Toxic Epidermal Necrolysis (TEN) patients. In vivoinjection of the blister fluid into epidermis of nude mice inducedmassive skin cell death, mimicking the human pathology of SJS/TEN. Chenet al. concluded that 15 kD granulysin mediated this undesired immuneresponse in the host. In contrast to the study by Chen et al., theinventors have determined that recombinant full-length 15 kD granulysinis not cytolytic and surprisingly exhibits substantially moreimmune-stimulating activity than 9 kD granulysin.

The inventors have also determined that 15 kD granulysin activatesmonocytes to differentiate into monocyte derived-dendritic cells(MO-DC), thereby initiating an immune response in a subject that canproduce allospecific T cells, and can therefore be used as a vaccineadjuvant, alone or in combination with other vaccine preparations ortherapeutic agents.

A method has also been developed to produce 15 kD granulysin in vitrousing a recombinant vector encoding full-length 15 kD granulysin. Inaddition, methods are disclosed for using 15 kD granulysin to activatedesired immune responses, in vaccination, infection, or otherimmunotherapies. Additionally, methods are disclosed for using the 15 kDgranulysin to block induction of an immune response in autoimmunediseases or organ transplantation.

The disclosure also relates to using 15 kD granulysin to induce thedifferentiation of monocytes into monocyte-derived dendritic cells. Inseveral embodiments, the methods are used to identify monocyte-deriveddendritic cells from other cells of the immune system, such asmacrophages. In a further embodiment, the methods are used to initiateor stimulate an immune response in a host following administration of 15kD granulysin to treat, lessen or inhibit an immune-based disorder.Alternatively, the method can be used for in vitro differentiation ofmonocytes into dendritic cells and/or production of allospecific Tcells. The methods are also effective for inhibiting an undesired immuneresponse in an immunocompromised host, for example someone who has or isa candidate for undergoing solid organ transplantation, such as adialysis patient.

In some embodiments, 15 kD granulysin can inhibit the development of atumor in a host, for example to treat or inhibit the tumor. In anotheraspect, 15 kD granulysin is effective for the treatment of anon-infectious disease or disorder. In another embodiment, 15 kDgranulysin may be used as a vaccine adjuvant, for example as ananti-tumor vaccine adjuvant, e.g., an adjuvant in a Hepatitis B virus(HBV) or Hepatitis C virus (HCV) prophylactic vaccine. In anotherembodiment, 15 kD granulysin can be used as an adjuvant for anallergen-based vaccine.

In one aspect, the immunogenicity of an antigen may be enhanced byincreasing the specific antigen presenting function of dendritic cellsin a mammalian host. Prior to immunization with an antigen, the host istreated with 15 kD granulysin. This activates and expands the number ofmonocytes in the host and causes the monocytes to differentiate intomonocyte-derived dendritic cells. In some instances, the host may begiven a local, e.g., subcutaneous, intramuscular, etc., injection ofantigen in combination with 15 kD granulysin, such as the administrationof an immunostimulatory sequence, for example a CpG motif containingoligonucleotide, interleukin-1 (IL-1), lipopolysaccharide (LPS), or anadditional toll-like receptor (TLR) agonist. In other examples, theantigen may be administered to a subject as a fusion protein with 15 kDgranulysin. The disclosed methods promote the recruitment and maturationof monocytes into monocyte-derived dendritic cells while concurrentlyinducing antigen-specific migration from the blood vessels to tissuesand subsequently the migration of monocyte-derived dendritic cells tolymphoid organs. The monocyte-derived dendritic cells can then interactwith and present processed antigens to local T cells that in turninitiate an immune response to the presented antigen.

The methods of the invention are particularly useful in subjects with asub-optimal immune response, for example in conditions of chronicinfection, a lack of immune response to tumor antigens, anergic orimmunosuppressed individuals, or a low responsiveness to allergens.

In one aspect, the disclosed methods are used to enhance the host'simmune response to tumor cells present in the host's body.

In another embodiment, 15 kD granulysin is used to delay development ofa tumor in a subject, induce tolerance to a transplanted organ in amammalian transplant recipient, or inhibit an immune-based disorder in asubject, such as an autoimmune disorder. A therapeutically effectiveamount of 15 kD granulysin can be administered to a subject having oneor more of these conditions.

In yet another embodiment, a method of generating an activated Tlymphocyte is provided, wherein a monocyte-derived dendritic cell isproduced following incubation with 15 kD granulysin, and themonocyte-derived dendritic cell is contacted with a T lymphocyte invitro, thereby producing an activated T lymphocyte. In other examples, amonocyte-derived dendritic cell is produced following incubation with afusion protein of 15 kD granulysin and a target antigen.

The foregoing and other features of the disclosure will become apparentfrom the following detailed description, which proceeds with referenceto the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1C show flow cytometry data demonstrating the activationactivity of 15 kD granulysin (10 nM) on human monocytes in vitro. FIG.1A data were obtained using human monocytes in cell culture medium inthe absence of granulysin. FIG. 1B and FIG. 1C show flow cytometry dataobtained from human monocytes incubated for 2 days with 9 kD granulysinor 15 kD granulysin, respectively (10 nM). FIG. 1C (15 kD granulysintreatment) shows an increase in both cell granularity and cell size incomparison to either 9 kD granulysin treatment or incubation in cellculture medium. Cell size is shown on the x-axis; cell granularity isshown on the y-axis.

FIG. 2A-2D are graphs of cell surface expression markers of CD14+ humanmonocytes after 2 days incubation with 15 kD granulysin (10 nM) or 9 kDgranulysin (10 nM). Cell surface expression phenotype data is shown forthe cell surface markers CD40 (FIG. 2A), CD83 (FIG. 2B), CD80 (FIG. 2C),and CD209 (FIG. 2D). Treatment of human CD14+ monocytes with 15 kDgranulysin (10 nM) was found to activate monocytes in vitro as shown byan up-regulation of dendritic cell specific surface markers, CD40, CD80,CD83, and CD209.

FIG. 3A-3F are graphs of cell surface expression markers of CD14+ humanmonocytes cultured with 15 kD granulysin (10 nM) and IL-4 for 5 days.Lipopolysaccharide (LPS) was added to the culture on day 5 to inducematuration of resulting monocyte-derived dendritic cells, and the cellswere analyzed on day 7 by Fluorescent Activated Cell Sorting (FACS).Cell surface expression phenotype data is shown for the markers CD86(FIG. 3A), CD209 (FIG. 3B), CD14 (FIG. 3C), CD11b (FIG. 3D), CD80 (FIG.3E), and HLA-DR (FIG. 3F). Incubation of human CD14+ monocytes in vitrowith 15 kD granulysin (10 nM), IL-4 and LPS resulted in thedifferentiation of monocytes into mature dendritic cells (derived frommonocytes) as shown by an up-regulation of dendritic cellspecific-surface markers and a down-regulation of monocyte specific cellsurface markers (e.g., CD14).

FIG. 3G shows a series graphs of cell surface expression markers thatillustrate 15 kD granulysin activates immature dendritic cells to becomemature dendritic cells. Elutriated human monocytes (2×10⁶/ml) wereincubated in RPMI-1640 supplemented with 10% FCS in the presence ofGM-CSF (10 ng/ml) and IL-4 (10 ng/ml). After 5 days, 15 kD granulysin(10 nM) was added and the culture continued for an additional 24 hours.Cells were harvested, stained with fluorescent antibodies, and analyzedby FACS.

FIG. 4A-4B are graphs of cell surface expression of CD14+ humanmonocytes cultured with GM-CSF in the presence or absence of IL-4. FIG.4A shows cell surface expression of marker, CD1a, on the surface ofmonocytes incubated in the presence of cell culture medium only, GM-CSF(10 ng/ml), or in the presence of 15 kD granulysin (10 nM). Cells wereharvested after five days, stained with APC-conjugated anti-human CD1aand analyzed by FACS. FIG. 4B shows the cell surface expression of CD1aon the surface of monocytes incubated under identical conditions asdescribed in FIG. 4, except that the monocytes were incubated for theduration of the experiment with 10 ng/ml interleukin-4 (IL-4). FIG. 4Aand FIG. 4B demonstrate that GM-CSF but not 15 kD granulysin inducesexpression of CD1a on the cell surface of monocytes.

FIG. 5A-5C are graphs demonstrating the fold-increase in cytokineexpression (IL-6, IL-1β or TNFα) upon administration of 15 kD granulysin(10 nM) to monocytes in vitro in the presence or absence of an pertussistoxin, an agent that inhibits signaling through G-protein coupledreceptors. FIG. 5A shows significant fold-increase of TNFα expressionwhen monocytes were incubated in the presence of 15 kD granulysin or inthe presence of 15 kD granulysin and pertussis toxin (100 ng/ml). mRNAwas obtained from the cultured monocytes and converted to cDNA.Quantitative PCR allowed for the calculation of fold-increase inexpression relative to a house-keeping gene. FIGS. 5B and 5C showresults of similar experiments for the fold-increase in cytokineexpression of IL-10 and IL-6, respectively.

FIG. 6 is a graph showing fold-stimulation of allospecific T cells uponincubation of human CD14+ monocytes with 15 kD granulysin or GM-CSF. Thefour upper rows of FIG. 6, monocytes were activated by incubation withGM-CSF (10 ng/ml) or GM-CSF and IL-4 (10 ng/ml). After 4 days,lipopolysaccharide (LPS) was added to the culture to induce dendriticcell maturation. Cells were harvested on day 6 and used to stimulateallogeneic T cells. After five additional days, cellular proliferationwas measured and reported as fold-stimulation above T-cells alone. Thefour lower rows of FIG. 6 demonstrate fold-stimulation of allospecific Tcells upon incubation of human CD14+ monocytes with 15 kD granulysin (10nM) or 15 kD granulysin and IL-4 (10 ng/ml). Identical to the upper rowsof FIG. 6, LPS was added to the cells at day 4 to induce dendritic cellmaturation. The cells were harvested on day 6 and used to stimulateallogeneic T cells. After five additional days, cellular proliferationwas measured and expressed as fold-stimulation above T-cells alone.

FIG. 7 is a series of graphs that show 15 kD granulysin induces Th1 andTh17 cell expansion, but inhibits Th2 cells. Peripheral bloodmononuclear cells (2×10⁶/ml) were incubated in RPMI-1640 supplementedwith 10% FCS. Where indicated, anti-CD3 antibody was added at 0.001μg/ml and 15 kD granulysin was added at 10 nM. After 7 days, cells wererestimulated with PMA (5 ng/ml) and ionomycin (500 ng/ml) for 1 hour atwhich time Golgistop was added and the incubation continued for another4 hours. Cells were harvested and stained for surface CD4 or CD8. Cellswere then fixed, permeabilized, and stained with fluorescent antibodiesspecific for IFNγ, IL-17 or IL-10 and analyzed by FACS.

FIGS. 8A-8C are a series of panels showing activation of human monocytesby 15 kD granulysin. FIG. 8A is a series of digital images showingpurified CD14⁺ monocytes cultured in medium alone (left), medium plus 10ng/ml GM-CSF (middle), or medium supplemented with 10 nM 15 kDgranulysin (right) for 6 hours. Images taken with a 10× objective. FIG.8B is a series of graphs showing expression of cell surface molecules bymonocytes cultured with medium, 15 kD granulysin (10 nM), or GM-CSF (10ng/ml) for 24 hours, then stained with fluorescent antibodies andanalyzed by flow cytometry. FIG. 8C is a series of plots showingexpression of IL-1β, IL-6, and TNFα in monocytes cultured with medium,15 kD granulysin (10 nM), or GM-CSF (10 ng/ml) for 24 hours, thenstained with fluorescent antibodies and analyzed by flow cytometry.

FIGS. 9A-9B are a series of graphs showing effect of 15 kD granulysin onmaturation of dendritic cells and activation of cytokine expression in Tcells. FIG. 9A is a series of graphs showing that 15 kD granulysininduces differentiation of immature dendritic cells to mature dendriticcells. Monocytes were cultured for 4 days with 10 ng/ml GM-CSF plus 10ng/ml IL-4, and then for another 24 hours with medium (gray) or 10 nM 15kD granulysin (black line). Cells were stained with fluorescentantibodies and analyzed by flow cytometry. FIG. 9B is a series of graphsshowing activation of cytokine expression in T cells cultured withdendritic cells induced with 15 kD granulysin. Purified T cells wereadded to allogeneic monocytes that had been cultured for 4 days withmedium, 10 nM 15 kD granulysin, or 10 ng/ml GM-CSF. After a 5 dayculture, cytokine production was measured by intracellular staining andflow cytometry.

FIGS. 10A-10C are a series of graphs showing the effect of granulysinexpression in mice challenged with CT26 tumor cells. Wild type orgranulysin transgenic mice (GNLY^(+/−)) were injected with CT26 tumorcells in the left flank, and tumor and draining lymph nodes were removedafter 12-14 days. FIG. 10A shows the weight of excised tumors. Eachpoint represents one animal; each panel represents one experiment.*p<0.01 FIG. 10B shows the percentage of the indicated cells afterstimulation of tumor infiltrating lymphocytes (TIL) stimulated in vitrowith PMA/ionomycin. Expression of TNFα and IFNγ was measured by flowcytometry. *p<0.01 FIG. 10C shows the numbers of CD40+ and CD86+ cellsin the tumor (TIL) and draining lymph nodes (LN). *p<0.01

SEQUENCE LISTING

Any nucleic acid and amino acid sequences listed herein or in theaccompanying sequence listing are shown using standard letterabbreviations for nucleotide bases, and three letter code for aminoacids, as defined in 37 C.F.R. 1.822. In at least some cases, only onestrand of each nucleic acid sequence is shown, but the complementarystrand is understood as included by any reference to the displayedstrand.

The Sequence Listing is submitted as an ASCII text file in the form ofthe file named Sequence_Listing.txt, which was created on Jun. 6, 2016,and is 3016 bytes, which is incorporated by reference herein.

SEQ ID NO: 1 is an exemplary 15 kD granulysin amino acid sequence.

SEQ ID NO: 2 is an exemplary 15 kD granulysin nucleic acid sequence.

DETAILED DESCRIPTION

The inventors have discovered that 9 and 15 kD granulysin have verydifferent activities. For example, 9 kD granulysin is cytotoxic while 15kD granulysin is not. Recombinant 9 kD granulysin lyses a wide varietyof tumor cells as well as pathogens, including gram positive and gramnegative bacteria, fungi, parasites and intracellular organisms such asM. tuberculosis. In contrast, 15 kD granulysin does not kill any cells(eukaryotic or prokaryotic).

The 15 kD but not 9 kD granulysin activates monocytes (FIGS. 1A-1C andFIGS. 2A-2D). Moreover, 15 kD but not 9 kD granulysin activatesmonocytes to become immature dendritic cells (FIGS. 3A-3F), and canactivate immature dendritic cells that had been activated by theconventional method (GM-CSF plus IL-4) to become mature dendritic cells(FIG. 3G). The 15 kD granulysin, but not GM-CSF, induces rapidphenotypic changes in monocytes (FIG. 8A).

Recombinant 15 kD granulysin or GM-CSF induces monocytes to expressCD1c, CD11a, CD29, CD40, CD54, CD80, CD86 and HLA-DR (see FIG. 3G).GM-CSF, but not 15 kD granulysin, induces expression of CD1a onmonocytes.

The 15 kD granulysin induces monocytes to express IL-1α, IL-1β, IL-6,IL-12, IL-23 and TNF-α, but does not induce expression of IL-10, IL-18,or IL-27. In addition, 15 kD granulysin causes a rapid gene expressionin monocytes, peaking at about 4 hours, but GM-CF induced geneexpression is not evident until about 24 hours.

Monocytes activated by 15 kD granulysin or GM-CSF activate allospecificT cells (FIG. 6), but 9 kD granulysin does not exhibit this activity.Incubation of monocytes with 15 kD granulysin causes rapid phenotypicchanges and a concomitant increase in expression of a panel ofproinflammatory cytokines. Lastly, addition of 15 kD granulysin tounseparated peripheral blood mononuclear cells induces both Th1 and Th17responses, but inhibits Th2 responses (FIG. 7). Thus, 15 kD granulysinis a potent (effective in the picomolar to nanomolar range) and novelactivator of the proinflammatory immune response that is believed to beuseful as a novel adjuvant for vaccines. These results also enable an invitro method of stimulating production of an allospecific T lymphocyte.

I. Abbreviations

APC: antigen presenting cell

CA IV: carbonic anhydrase isozyme IV

DC: dendritic cell

FACS: fluorescent activated cell sorting

Flt-3L: flt-3 ligand

GM-CSF: granulocyte macrophage colony stimulating factor

G-CSF: granulocyte colony stimulating factor

HGF: hepatocyte growth factor

IFN-α: interferon alpha

IFN-γ: interferon gamma

IL: interleukin

kD: kilodalton

LPS: lipopolysaccharide

M-CSF: macrophage colony stimulating factor

mDC: myeloid dendritic cell

MO: monocyte

MO-DC: monocyte-derived dendritic cell

pDC: plasmacytoid dendritic cell

TIL: tumor-infiltrating lymphocytes

TLR: toll-like receptors

TSLP: thymic stromal lymphopoietin

VIP: vasoactive intestinal peptide

II. Explanation of Terms

It is to be understood that the present disclosure is not limited to theparticular methodology, protocols, cell lines, animal species or genera,constructs and reagents described, as such may vary. It is alsounderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present disclosure.

As used herein the singular forms “a,” “and,” and “the” refer to boththe singular as well plural, unless the context clearly indicatesotherwise. For example, reference to “an immunization” includes aplurality of such immunizations and reference to “the cell” includesreference to one or more cells and equivalents thereof known to one ofordinary skill in the art, and so forth. As used herein the term“comprises” means “includes.” Thus, “a composition comprising 15 kDgranulysin” means “including 15 kD granulysin” without excluding otheradditional components.

It is further to be understood that all base sizes or amino acid sizes,and all molecular weight or molecular mass values, given for nucleicacids or polypeptides are approximate, and are provided for descriptivepurposes, unless otherwise indicated. Although many methods andmaterials similar or equivalent to those described herein can be used,particular suitable methods and materials are described below. In caseof conflict, the present specification, including explanation of terms,will control.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

All of the technical and scientific terms used herein have the samemeaning as commonly understood to one of ordinary skill in the art towhich this invention belongs unless clearly indicated otherwise.

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Activation Agent: refers to a compound, such as a naturally occurringprotein, which acts on monocytes to expand (e.g., proliferate) anddifferentiate into monocyte-derived dendritic cells (MO-DC). In apreferred embodiment, the activating agent is 15 kD granulysin. The doseof the activating agent will be effective to substantially increase thenumber of monocytes. The increase in the number of monocytes afteractivation can be quite high, usually by at least 2-fold more, typically5-fold more, and may be as high as about 20- to about 75-fold more.Monocytes activated by 15 kD granulysin can differentiate intomonocyte-derived dendritic cells and will therefore typically expressincreased levels of CD40, CD80, and CD83 as compared to non-activatedmonocytes. Additionally, monocytes activated through the administrationof 15 kD granulysin and thus differentiated into monocyte-deriveddendritic cells will express reduced levels of lineage markers such asCD14, as compared to untreated monocytes, and can be identified on thebasis of these criteria, among others.

Allergen/Allergy: is a disorder of the immune system also referred to asatopy. Allergic reactions occur to normally harmless environmentalsubstances known as allergens e.g., dust mite dander. Common allergicreactions include eczema, hives, hay fever, asthma, and food and drugallergies. In some instances, a subject's immune response to an allergenis severe enough to induce anaphylactic shock.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous immunogens. The term “antigen”includes all related antigenic epitopes. Examples of exemplary antigensof interest include proteins, polypeptides, polysaccharides, a DNAmolecule, a RNA molecule, a whole cell lysate, an apoptotic cell, or acombination thereof.

An “antigenic polypeptide” is a polypeptide to which an immune response,such as a T cell response or an antibody response, can be stimulated.“Epitope” or “antigenic determinant” refers to a site on an antigen towhich B and/or T cells respond. T cells can respond to the epitope whenthe epitope is presented in conjunction with an MHC molecule. Epitopescan be formed both from contiguous amino acids (linear) or noncontiguousamino acids juxtaposed by tertiary folding of an antigenic polypeptide(conformational). Epitopes formed from contiguous amino acids aretypically retained on exposure to denaturing solvents whereas epitopesformed by tertiary folding are typically lost on treatment withdenaturing solvents. Normally, a B-cell epitope will include at leastabout 5 amino acids but can be as small as 3-4 amino acids. A T-cellepitope, such as a CTL epitope, will include at least about 7-9 aminoacids, and a helper T-cell epitope at least about 12-20 amino acids.Normally, an epitope will include between about 5 and 15 amino acids,such as, 9, 10, 12 or 15 amino acids. The amino acids are in a uniquespatial conformation. In one particular example, the antigen is anantigen obtained from a subject who is a donor, such as of an organ orof bone marrow, to another genetically different individual. In anotherexample, the antigen is a tumor antigen.

A “target antigen” includes, but is not limited to, an antigen that ispresent in a disease or disorder, such as a tumor antigen, an autoimmuneantigen, an allergen antigen, or an antigen expressed in solid organtransplantation rejection. A target antigen may be any antigen for whichit is desirable to modulate an immune response in a subject. Inparticular embodiments, the disclosed methods enhance or stimulate animmune response in a subject against a target antigen (for example, atumor antigen). In other embodiments, the disclosed methods inhibit ordecrease an immune response in a subject against a target antigen (forexample, an autoimmune antigen, an allergen, or an antigen expressed insolid organ transplantation rejection).

Autoimmune disorder: A disorder or disease in which the immune systemproduces an immune response (e.g. a B cell or a T cell response) againstan endogenous antigen, with consequent injury to tissues. The injury maybe localized to certain organs, such as thyroiditis, or may involve aparticular tissue at different locations, such as Goodpasture's disease,or may be systemic, such as lupus erythematosus.

Cancer: A malignant neoplasm that has undergone characteristic anaplasiawith loss of differentiation, increased rate of growth, invasion ofsurrounding tissue, and is capable of metastasis. For example, thyroidcancer is a malignant neoplasm that arises in or from thyroid tissue,and breast cancer is a malignant neoplasm that arises in or from breasttissue (such as a ductal carcinoma). Residual cancer is cancer thatremains in a subject after any form of treatment given to the subject toreduce or eradicate the cancer. Metastatic cancer is a cancer at one ormore sites in the body other than the site of origin of the original(primary) cancer from which the metastatic cancer is derived.

Chemokine (chemoattractant cytokine): A type of cytokine (a solublemolecule that a cell produces to control reactions between other cells)that specifically alters the behavior of leukocytes (white blood cells).Examples include, but are not limited to, interleukin 8 (IL-8), plateletfactor 4, melanoma growth stimulatory protein, and the like.

Chemotherapy; chemotherapeutic agents: As used herein, any chemicalagent with therapeutic usefulness in the treatment of diseasescharacterized by abnormal cell growth. Such diseases include tumors,neoplasms, and cancer as well as diseases characterized by hyperplasticgrowth such as psoriasis. In one embodiment, a chemotherapeutic agent isan agent of use in treating neoplasms such as solid tumors. In oneembodiment, a chemotherapeutic agent is a radioactive molecule. One ofskill in the art can readily identify a chemotherapeutic agent of use(e.g. see Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 inHarrison's Principles of Internal Medicine, 14th edition; Perry et al.,Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2^(nd) ed., © 2000Churchill Livingstone, Inc; Baltzer L, Berkery R (eds): Oncology PocketGuide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; FischerD S, Knobf M F, Durivage H J (eds): The Cancer Chemotherapy Handbook,4th ed. St. Louis, Mosby-Year Book, 1993).

Contacting: Placement in direct physical association, including both asolid and liquid form. Contacting can occur in vitro with isolated cellsor in vivo by administering to a subject.

Cytokine: Proteins made by cells that affect the behavior of othercells, such as lymphocytes. In one embodiment, a cytokine is achemokine, a molecule that affects cellular trafficking. The term“cytokine” is used as a generic name for a diverse group of solubleproteins and peptides that act as humoral regulators at nanomolar topicomolar concentrations and which, either under normal or pathologicalconditions, modulate the functional activities of individual cells andtissues. These proteins also mediate interactions between cells directlyand regulate processes taking place in the extracellular environment.Examples of cytokines include, but are not limited to, tumor necrosisfactor α (TNFα), interleukin-6 (IL-6), interleukin-10 (IL-10),interleukin-12 (IL-12), macrophage inflammatory protein 2 (MIP-2),keratinocyte derived cytokine (KC), and interferon-γ (INF-γ)

Decrease: Becoming less or smaller, as in number, amount, size, orintensity. In one example, decreasing the risk of a disease (such as fortumor formation) includes a decrease in the likelihood of developing thedisease by at least about 20%, for example by at least about 30%, 40%,50%, 60%, 70%, 80%, or 90%. In another example, decreasing the risk of adisease includes a delay in the development of the disease, for examplea delay of at least about six months, such as about one year, such asabout two years, about five years, or about ten years.

In one example, decreasing the signs and symptoms of a tumor includesdecreasing the size, volume, tumor burden or number of tumors (such asskin tumors) or metastases by a desired amount, for example by at leastabout 5%, 10%, 15%, 20%, 25%, 30%, 50%, 75%, or even at least about 90%,as compared to a response in the absence of the therapeutic composition.

Dendritic cells (DC): Dendritic cells are antigen presenting cells (APC)involved in immune responses. Dendritic cells include plasmacytoiddendritic cells and myeloid dendritic cells. Their major function is toobtain antigen in tissues, migrate to lymphoid organs and present theantigen in order to activate local T cells, which in turn generate animmune response. Immature dendritic cells originate in the bone marrowand reside in the periphery as immature cells. In one embodiment, adendritic cell is a plasmacytoid dendritic cell. Plasmacytoid dendriticcells differentiate from precursors called “DC2” while myeloid dendriticcells differentiate from precursors termed “DC1.”

Dendritic cells are capable of evolving from immature, antigen-capturingcells to mature, antigen-presenting T cells; converting antigens intoimmunogens and expressing molecules such as cytokines, chemokines,co-stimulatory molecules and proteases to initiate an immune response.

Dendritic cells are derived from hematopoietic stem cells in the bonemarrow and are widely distributed as immature cells within all tissues,particularly those that interface with the environment (e.g. skin,mucosal surfaces, etc.) and in lymphoid organs. Immature dendritic cellsare recruited to sites of inflammation in peripheral tissues followingpathogen or foreign-body invasion. “Immature” dendritic cells mayexpress the chemokine receptors CCR1, CCR2, CCR5, CCR6 and CXCR1.Immature dendritic cells capture antigens by phagocytosis,macropinocytosis or via interaction with a variety of cell surfacereceptors and endocytosis. Internalization of foreign antigens cansubsequently trigger their maturation and migration from peripheraltissues to lymphoid organs (see below).

The ability of dendritic cells to regulate immunity is dependent ondendritic cell differentiation, as it depends on their maturation state.A variety of factors can induce differentiation following antigen uptakeand processing within dendritic cells, including: whole bacteria orbacterial-derived antigens (e.g. lipopolysaccharide), inflammatorycytokines, ligation of select cell surface receptors (e.g. CD40) andviral products (e.g. double-stranded RNA). During their conversion fromimmature to mature cells, dendritic cells undergo a number of phenotypeand functional changes. The process of dendritic cell maturation, ingeneral, involves a redistribution of major histocompatibility complex(MHC) molecules from intracellular endocytic compartments to thedendritic cell surface, down-regulation of antigen internalization, anincrease in the surface expression of co-stimulatory molecules,morphological changes (e.g. formation of dendrites), cytoskeletonre-organization, secretion of chemokines, cytokines and proteases, andsurface expression of adhesion molecules and chemokine receptors.Dendritic cells are characterized by their distinctive morphology andhigh levels of surface MHC-class II expression, such as CD40 and CD80markers.

Dendritic Cell Precursor: Immature cells that can differentiate intodendritic cells. In one embodiment a dendritic cell precursor is a DC1cell that differentiates into myeloid cells (e.g. a monocyte).

Differentiation: The process by which cells become more specialized toperform biological functions, and differentiation is a property that istotally or partially lost by cells that have undergone malignanttransformation. For example, dendritic cell precursors such as monocytesdifferentiate into dendritic cells under the influence of certaincytokines and growth factors.

Epitope: An antigenic determinant. These are particular chemical groupsor peptide sequences on a molecule that are antigenic, e.g., that elicita specific immune response. An antibody binds a particular antigenicepitope.

Expansion and Activation: Refers to the length of time required foractivation and expansion of monocytes into monocyte derived-dendriticcells. The time taken is usually at least about 2 days, more usuallyabout 1 week, and may take about 10 days to about 2 weeks for optimalexpansion. The length of time allotted for activation and expansion canbe predicted based on previous trials with the activation agent at asimilar dose, or may be monitored individually by quantitating thechange in the number of dendritic cells present in the peripheral bloodof a subject.

Flt-3 ligand (flt-3L): A factor that binds to the flt-3 receptor. Theflt-3 ligand promotes long-term expansion and differentiation of humanpro-B-cells in the presence of IL-7, or IL-7 and IL-3. The flt-3 ligandis known to support the survival of precursor cell types in the lineageof blood-forming cells, such as highly proliferative potential colonyforming cells (e.g. see Lyman et al., Cell 75:1157-67, 1993).

Granulocyte/macrophage colony-stimulating factor (GM-CSF): A factorwhich modulates the maturation and function of dendritic cells,(Witmer-Pack et al., J. Exp. Med. 166:1484-98, 1987).

GM-CSF is a monomeric protein of 127 amino acids with two glycosylationsites. The protein is synthesized as a precursor of 144 amino acids,which included a hydrophobic secretory signal sequence at theamino-terminal end. The human gene has a length of approximately 2.5kilobase (kb) and contains four exons. The distance between the GM-CSFgene and the IL-3 gene is approximately 9 kb. The human GM-CSF gene mapsto chromosome 5q22-31.

GM-CSF was isolated initially as a factor stimulating the growth ofmacrophage/granulocyte-containing colonies in soft agar cultures. GM-CSFis also involved in the growth and development of granulocyte andmacrophage progenitor cells. GM-CSF stimulates myeloblasts andmonoblasts and triggers irreversible differentiation of these cells.GM-CSF synergizes with erythropoietin in the proliferation of erythroidand megakaryocytic progenitor cells.

GM-CSF has been used clinically for the physiological reconstitution ofhematopoiesis in diseases characterized either by an aberrant maturationof blood cells or by a reduced production of leukocytes. The usual dose,route and schedules for GM-CSF are 5-10 μg/kg/day either by 4-6 hoursintravenous infusion or by subcutaneous injection.

Granulysin: Granulysin is expressed from a gene located on humanchromosome 2 and comprises 6 exons within a 3.9 kb genomic locusencoding at least four alternatively spliced transcripts (NKG5, 519, 520and 522). The predicted amino acid sequence of transcript 519 can befound in U.S. Pat. No. 4,994,369 (incorporated herein by reference).Granulysin is a cationic molecule present in the granules of cytotoxic Tcells and NK cells. Granulysin is expressed as a 15 kD naturallyoccurring precursor protein, known as 15 kD granulysin. Granulysin isconstitutively secreted as the 15 kD precursor form, a portion of whichis localized in cytolytic granules where it is post-translationallyprocessed into a 9 kD form. Granulysin in the 9 kD form is known toexhibit potent cytotoxic activity against a broad panel of microbialtargets, including transplant cells, bacteria, fungi, and parasites(Stenger et al., Immunol. Today 20:390-394, 1999; Clayberger andKrensky, Curr. Opin. Immunol. 15:560-565, 2003; Hanson et al., Mol.Immunol. 36:413-422, 1999; Sarwal et al., Hum. Immunol. 62:21-31, 2001;Wang et al., J. Immunol. 165:1486-1490, 2000), and damaging negativelycharged cell membranes because of its positive charge (Kaspar et al., J.Immunol. 167:350-356, 2001). Deng et al. (J. Immunol. 174:5243-5248,2005) also observed that the 9 kD post-translational form of granulysinpossessed anti-microbial activity and chemotactic activity.

Until recently, full-length recombinant 15 kD granulysin had not beensuccessfully isolated or characterized. Animal models are difficult toprepare because mice do not possess the granulysin gene.

As referred to herein, 15 kD granulysin refers to the full-lengthprecursor form of granulysin with a molecular weight of about 15kilodaltons (and is substantially free of 9 kD granulysin). The instantinvention is distinct from the previously identified and characterizedform of 9 kD granulysin (referred to in the art, and herein, as “9 kDgranulysin”) which has an approximate molecular weight of about 9,000daltons. In a preferred embodiment, 15 kD granulysin includes SEQ IDNO. 1. In additional embodiments, 15 kD granulysin includes SEQ ID NO:2, a nucleic acid sequence encoding 15 kD granulysin. In someembodiments of the disclosed methods and compositions, the 15 kDgranulysin includes peptides that have at least 95%, at least 98%, or atleast 99% sequence identity to SEQ ID NO:1, and retain the describedactivity of SEQ ID NO: 1. Alternatively, one, two or three conservativesubstitutions can be made to SEQ ID NO: 1. In other embodiments, the 15kD granulysin includes nucleic acid molecules that have at least 85%, atleast 90%, at least 95%, at least 98%, or at least 99% sequence identityto SEQ ID NO: 2 and encode a polypeptide that retains the describedactivity of 15 kD granulysin.

Nucleic acid and protein sequences for 15 kD granulysin are publiclyavailable. For example, GENBANK® Accession No. NM_012483 discloses anexemplary 15 kD granulysin nucleic acid sequence, and GENBANK® AccessionNo. NP_036615 discloses an exemplary 15 kD granulysin amino acidsequence, both of which are incorporated by reference as provided byGENBANK® on Oct. 8, 2010.

Immune response: A response of a cell of the immune system, such as a Bcell, or a T cell to a stimulus. In one embodiment, the response isspecific for a particular antigen (an “antigen-specific response”). Inanother embodiment, the response is an inflammatory response.

A “parameter of an immune response” is any particular measurable aspectof an immune response, including, but not limited to, cytokine secretion(IL-6, IL-10, IFN-γ, etc.), immunoglobulin production, dendritic cellmaturation, and proliferation of a cell of the immune system. One ofskill in the art can readily determine an increase in any one of theseparameters, using known laboratory assays. In one specific non-limitingexample, to assess cell proliferation, incorporation of ³H-thymidine canbe assessed. A “substantial” increase in a parameter of the immuneresponse is a significant increase in this parameter as compared to acontrol. Specific, non-limiting examples of a substantial increase areat least about a 50% increase, at least about a 75% increase, at leastabout a 90% increase, at least about a 100% increase, at least about a200% increase, at least about a 300% increase, and at least about a 500%increase. One of skill in the art can readily identify a significantincrease using known statistical methods.

Immunocompromised: An immunocompromised subject is a subject who isincapable of developing or unlikely to develop a robust immune response,usually as a result of disease, malnutrition, or immunosuppressivetherapy. An immunocompromised immune system is an immune system that isfunctioning below normal. Immunocompromised subjects are moresusceptible to opportunistic infections, for example viral, fungal,protozoan, or bacterial infections, prion diseases, and certainneoplasms. Those who can be considered to be immunocompromised include,but are not limited to, subjects with AIDS (or HIV positive), subjectswith severe combined immune deficiency (SCID), diabetics, subjects whohave had transplants and who are taking immunosuppressives, and thosewho are receiving chemotherapy for cancer. Immunocompromised individualsalso includes subjects with most forms of cancer (other than skincancer), sickle cell anemia, cystic fibrosis, those who do not have aspleen, subjects with end stage kidney disease (dialysis), and those whohave been taking corticosteroids on a frequent basis by pill orinjection within the last year. Subjects with severe liver, lung, orheart disease also may be immunocompromised.

Immunostimulatory CpG motifs: Immunostimulatory sequences that triggermonocytes, macrophages and lymphocytes to produce a variety ofpro-inflammatory cytokines and chemokines. CpG motifs are found inbacterial DNA. The innate immune response elicited by CpG DNA reduceshost susceptibility to infectious pathogens, and can also triggerdetrimental inflammatory reactions. Immunostimulatory CpG motifs arefound in “D” and “K” type oligodeoxynucleotides (see, for example PCTPublication No. WO 01/51500, published on Jul. 19, 2001).

Interferon alpha (IFN-α): At least 23 different variants of IFN-α areknown. The individual proteins have molecular masses between 19-26 kDand consist of proteins with lengths of 156-166 and 172 amino acids. AllIFN-α subtypes possess a common conserved sequence region between aminoacid positions 115-151 while the amino-terminal ends are variable. ManyIFN-α subtypes differ in their sequences at only one or two positions.Naturally occurring variants also include proteins truncated by 10 aminoacids at the carboxyl-terminal end.

There are at least 23 different IFN-α genes. They have a length of 1-2kb and are clustered on human chromosome 9p22. Based upon the structurestwo types of IFN-alpha genes, designated class I and II, aredistinguished. They encode proteins of 156-166 and 172 amino acids,respectively.

Interferon gamma (IFN-γ): IFN-γ is a dimeric protein with subunits of146 amino acids. The protein is glycosylated at two sites, and the pI is8.3-8.5. IFN-γ is synthesized as a precursor protein of 166 amino acidsincluding a secretory signal sequence of 23 amino acids. Two molecularforms of the biologically active protein of 20 and 25 kD have beendescribed. Both of them are glycosylated at position 25. The 25 kD formis also glycosylated at position 97. The observed differences of naturalIFN-γ with respect to molecular mass and charge are due to variableglycosylation patterns. 40-60 kD forms observed under non-denaturingconditions are dimers and tetramers of IFN-γ. The human gene has alength of approximately 6 kb. It contains four exons and maps tochromosome 12q24.1.

Interleukin-2: IL-2 is a cytokine having a length of 133 amino acids.IL-2 has been approved by the Food and Drug Administration (FDA) for thetreatment of some forms of cancer, including kidney cancer, melanoma,and lymphoma. IL-2 can be administered via intravenous or subcutaneousinjections, where IL-2 is typically administered daily, or twice daily,over a course of several days, until the course of treatment iscomplete. IL-2 functions as an immune modulator, and stimulates theproliferation and activation of immune cells such as T cells and NaturalKiller cells.

Interleukin-4: The gene for Interleukin-4 (IL-4) is located onchromosome 5 at position q31. The nucleotide sequence of IL-4 wasisolated in 1986 and confirmed its similarity to the mouse protein,B-Cell Stimulating Factor (BCSF-1). IL-4 is a cytokine thatdifferentiates naïve helper T cells into Th2 cells. IL-4 stimulates theproduction of IgE and induces eosinophil-mediated attacks againsthelminthic infections and allergens. IL-4 is currently used fortherapeutic intervention in a wide range of malignant diseases as ananti-tumor agent

Interleukin-10: IL-10 is a homodimeric protein with subunits having alength of 160 amino acids that is a cytokine. Human IL-10 is a cytokinewith 73 percent amino acid homology to murine IL-10. The human IL-10gene contains four exons. IL10 inhibits the synthesis of a number ofcytokines such as IL-2 and IFN-γ in Th1 subpopulations of T cells butnot of Th2. IL10 can be detected with an ELISA assay. In addition, themurine mast cell line D36 can be used to bioassay human IL10. Theintracellular factor can also be detected by flow cytometry.

Isolated: An “isolated” biological component (such as a nucleic acid,peptide or protein) has been substantially separated, produced apartfrom, or purified away from other biological components in the cell ofthe organism in which the component naturally occurs, e.g., otherchromosomal and extrachromosomal DNA and RNA, and proteins. Nucleicacids, peptides and proteins which have been “isolated” thus includenucleic acids and proteins purified by standard purification methods.The term also embraces nucleic acids, peptides and proteins prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acids.

Leukocyte: Cells in the blood, also termed “white cells,” that areinvolved in defending the body against infective organisms and foreignsubstances. Leukocytes are produced in the bone marrow. There are 5 maintypes of white blood cell, subdivided between 2 main groups:polymorphonuclear leukocytes (neutrophils, eosinophils, basophils) andmononuclear leukocytes (monocytes and lymphocytes). When an infection ispresent, the production of leukocytes increases.

Mammal: This term includes both human and non-human mammals. Similarly,the term “subject” includes both human and veterinary subjects.

Maturation: The process in which an immature cell, such as an immaturedendritic cell, changes in form or function to become a functionallymature dendritic cell.

Neoplasm: An abnormal cellular proliferation, which includes benign andmalignant tumors, as well as other proliferative disorders.

Nucleic acid: A deoxyribonucleotide or ribonucleotide polymer in eithersingle or double stranded form, and unless otherwise limited,encompasses known analogues of natural nucleotides that hybridize tonucleic acids in a manner similar to naturally occurring nucleotides.

Parenteral: Administered outside of the intestine, e.g., not via thealimentary tract. Generally, parenteral formulations are those that willbe administered through any possible mode except ingestion. This termespecially refers to injections, whether administered intravenously,intrathecally, intramuscularly, intraperitoneally, intraarticularly, orsubcutaneously, and various surface applications including intranasal,intradermal, and topical application, for instance.

Pharmaceutical agent or drug: A chemical compound or composition capableof inducing a desired therapeutic or prophylactic effect when properlyadministered to a subject. Pharmaceutical agents include, but are notlimited to, chemotherapeutic agents and anti-infective agents.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful in this invention are conventional. Remington: TheScience and Practice of Pharmacy, The University of the Sciences inPhiladelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia,Pa., 21^(st) Edition (2005), describes compositions and formulationssuitable for pharmaceutical delivery of the proteins herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Inhibiting or treating a disease: “Inhibiting” a disease refers toinhibiting the full development of a disease, for example in a personwho is known to have a predisposition to a disease such as an autoimmunedisorder. An example of a person with a known predisposition is someonewith a history of familial cancers, or who has been exposed to factorsthat predispose the subject to a condition, such smoking or occupationalexposure to a carcinogen. Inhibition of a disease can span the spectrumfrom partial inhibition to substantially complete inhibition(prevention) of the disease. In some examples, the term “inhibiting”refers to reducing or delaying the onset or progression of a disease. Asubject to be administered with a therapeutically effective amount ofthe pharmaceutical compound to inhibit or treat the above illnesses canbe identified by standard diagnosing techniques for such a disorder, forexample, basis of family history, or risk factor to develop the diseaseor disorder. In contrast, “treatment” refers to a therapeuticintervention that ameliorates a sign or symptom of a disease orpathological condition after it has begun to develop.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified peptidepreparation is one in which the peptide or protein is more enriched thanthe peptide or protein is in its natural environment within a cell.Preferably, a preparation is purified such that the protein or peptiderepresents at least 50% of the total peptide or protein content of thepreparation. In some embodiments, a purified preparation contains atleast 60%, at least 70%, at least 80%, at least 85%, at least 90%, atleast 95% or more of the protein or peptide.

Subject at Risk: An individual, such as a human or a veterinary subject,that is prone to developing certain conditions, such as a tumor. Thiscan be due to their age, genotype, or due to an environmental exposure.Examples are a human subject who is exposed to a carcinogen due to anoccupational exposure, or a human subject exposed to cigarette smoke,either because that individual smokes or due to exposure to second-handsmoke, or a subject exposed to ultraviolet light, such as due totanning, or a subject genetically pre-disposed to developing a tumor.

T cell or T lymphocyte: A white blood cell critical to the immuneresponse. T cells include, but are not limited to, CD4⁺ T cells and CD8⁺T cells. A CD4⁺ T lymphocyte is an immune cell that carries a marker onits surface known as “cluster of differentiation 4” (CD4). These cells,also known as helper T cells, help orchestrate the immune response,including antibody responses as well as killer T cell responses. CD8⁺ Tcells carry the “cluster of differentiation 8” (CD8) marker. In oneembodiment, a CD8 T cell is a cytotoxic T lymphocyte. In anotherembodiment, a CD8 cell is a suppressor T cell.

As used herein, “allogeneic” encompasses a genetically differentphenotype present in non-identical individuals of the same species.Cells, tissues, organs, and the like from, or derived from, anon-identical individual of the same species are “allogeneic.” An“alloantigen” encompasses any antigen recognized by differentindividuals of the same species. Organisms, cells, tissues, organs, andthe like from, or derived from, a single individual, or from agenetically identical individual are “autologous.”

Therapeutic agent: Used in a generic sense, it includes treating agents,prophylactic agents, and replacement agents.

Therapeutically effective dose or amount: A dose or quantity of aspecified compound sufficient to inhibit advancement, or to causeregression of the disease, or which is capable of relieving symptomscaused by the disease, such as pain or swelling. For instance, this canbe the amount or dose of composition required to inhibit a tumor, delaythe development of a tumor, or reduce the risk of developing a tumor. Inone embodiment, a therapeutically effective amount of the composition isthe amount that alone, or together with one or more additionaltherapeutic agents (such as additional anti-neoplastic agents orimmunosuppressive agents), induces the desired response, such asinhibition or treatment of a tumor, such as skin cancer. In otherexamples, it is an amount of the composition that can cause regressionof an existing tumor, or treat one or more signs or symptoms associatedwith a tumor, in a subject. The preparations disclosed herein areadministered in therapeutically effective amounts.

In one example, a desired response is to inhibit, and in some examplesprevent, the development of a tumor. In another example, a desiredresponse is to delay the development, progression, or metastasis of atumor, for example, by at least about 3 months, at least about sixmonths, at least about one year, at least about two years, at leastabout five years, or at least about ten years. In a further example, adesired response is to decrease the occurrence of cancer, such asmelanoma, colon cancer, liver cancer or lung cancer. In another example,a desired response is to decrease the signs and symptoms of cancer, suchas the size, volume, or number of tumors or metastases. For example, thecomposition including 15 kD granulysin can, in some examples, decreasethe size, volume, tumor burden or number of tumors (such as colorectaltumors) by a desired amount, for example by at least 5%, at least 10%,at least 15%, at least 20%, at least 25%, at least 30%, at least 50%, atleast 75%, or even at least 90%, as compared to a response in theabsence of the therapeutic composition.

The effective amount of 15 kD granulysin that is administered to a humanor veterinary subject will vary depending upon a number of factorsassociated with that subject, for example the overall health of thesubject. An effective amount of an agent can be determined by varyingthe dosage of the product and measuring the resulting therapeuticresponse, such as the regression of a tumor. Effective amounts also canbe determined through various in vitro, in vivo or in situ,immunoassays. The disclosed agents can be administered in a single dose,or in several doses, as needed to obtain the desired response. However,the effective amount can be dependent on the source applied, the subjectbeing treated, the severity and type of the condition being treated, andthe manner of administration.

A therapeutically effective amount of 15 kD granulysin can beadministered systemically or locally. In addition, an effective amountof 15 kD granulysin can be administered in a single dose, or in severaldoses, for example daily, during a course of treatment. For example, atherapeutically effective amount of 15 kD granulysin can vary from about0.01 mg/kg body weight to about 1 g/kg body weight in some specific,non-limiting examples, or from about 0.01 mg/kg to about 60 mg/kg ofbody weight, based on efficacy.

The compositions disclosed herein have equal applications in medical andveterinary settings. Therefore, the general term “subject” is understoodto include all animals with a granulysin gene, including, but notlimited to, humans or veterinary subjects, such as other non-humanprimates, dogs, cats, horses, pigs, cows, and transgenic mice.

Toll-Like Receptors: Toll-Like Receptors (TLRs) are a class of proteinsthat play an important role in the innate immune response. They arereceptors that recognize structurally conserved molecules derived frompathogens. Upon entry of a pathogen derived molecule into a host, suchas via the lungs or skin, the TLRs activate a host's immune cellresponses. TLRs are a type of pattern recognition receptor (PRR) andrecognize molecules that are broadly shared among pathogens but aredistinguishable from the host, collectively referred to as, pathogenassociated molecular patterns (PAMPs). It has been estimated that mostmammalian species have between ten and fifteen types of TLRs. At leastthirteen TLRs (named TLR1 to TLR13) have been identified in humans andmice together, and equivalent forms of many of these have been found inother mammalian species. However, equivalents of certain TLRs found inhumans are not present in all mammals. For example, a gene coding for aprotein analogous to TLR10 in humans is present in mice, but appears tohave been damaged at some point in the past by a retrovirus. On theother hand, mice express TLRs 11, 12, and 13, none of which arerepresented in humans. Examples of molecules that can act as TLRagonists include flagellin, zymosan, poly (I:C), CpG oligonucleotides,endotoxins, resiquimod, imiquimod, gardiquimod, and lipopolysaccharide(LPS).

Tumor: An abnormal growth of cells, which can be benign or malignant.Cancer is a malignant tumor, which is characterized by abnormal oruncontrolled cell growth. Other features often associated withmalignancy include metastasis, interference with the normal functioningof neighboring cells, release of cytokines or other secretory productsat abnormal levels and suppression or aggravation of inflammatory orimmunological response, invasion of surrounding or distant tissues ororgans, such as lymph nodes, etc. “Metastatic disease” refers to cancercells that have left the original tumor site and migrate to other partsof the body for example via the blood vessels or lymph system.

The amount of a tumor in an individual is the “tumor burden” which canbe measured as the number, volume, or weight of the tumor. A tumor thatdoes not metastasize is referred to as “benign.” A tumor that invadesthe surrounding tissue and/or can metastasize is referred to as“malignant.” Examples of hematological tumors include leukemias,including acute leukemias (such as 11q23-positive acute leukemia, acutelymphocytic leukemia, acute myelocytic leukemia, acute myelogenousleukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic anderythroleukemia), chronic leukemias (such as chronic myelocytic(granulocytic) leukemia, chronic myelogenous leukemia, and chroniclymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease,non-Hodgkin's lymphoma (indolent and high grade forms), multiplemyeloma, Waldenstrom's macroglobulinemia, heavy chain disease,myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

Examples of solid tumors, such as sarcomas and carcinomas, includefibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy,pancreatic cancer, breast cancer (including basal breast carcinoma,ductal carcinoma and lobular breast carcinoma), lung cancers, ovariancancer, prostate cancer, hepatocellular carcinoma, squamous cellcarcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,medullary thyroid carcinoma, papillary thyroid carcinoma,pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma,renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladdercarcinoma, and CNS tumors (such as a glioma, astrocytoma,medulloblastoma, craniopharyrgioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, and retinoblastoma).

In several examples, a tumor is melanoma, esophageal cancer, livercancer, gastrointestinal cancer, colon cancer, or lung carcinoma. Inanother example, a tumor is a skin tumor.

Vaccine: As defined herein, a vaccine may be an immunogenic compositionfor stimulating an immune response against a target antigen. Suchcompositions may include a preparation of attenuated microorganisms(including but not limited to bacteria and viruses), livingmicroorganisms, killed microorganisms, antigens (including but notlimited to tumor antigens), polypeptides, nucleic acids, or vectorsencoding antigens, administered for the inhibition, amelioration ortreatment of non-infectious diseases, allergies, and tumors.

III. Overview of Several Embodiments

A method is provided for the enhancement of T cell mediated immuneresponses. In one embodiment, the method provides for the activation ofmonocytes, through the administration of 15 kD granulysin.

In another embodiment, the method provides for the differentiation ofmonocytes into monocyte-derived dendritic cells, thereby stimulating animmune response or up-regulating and already activated immune responsein a host.

In yet another embodiment, 15 kD granulysin can be used to treat orinhibit disease, or treat the symptoms of disease, such as an autoimmunedisorder or a tumor. In another aspect, 15 kD granulysin can be used asan adjuvant for a vaccine, such as a bacterial or viral vaccine.

In one embodiment, a target antigen is delivered to peripheral tissuesin combination with monocyte-derived dendritic cells, and may be givenas a combined formulation, or as a separate formulation. The antigen maybe further provided in a booster dose, in combination with otheradjuvants as is known in the art.

On maturation, the monocyte-derived dendritic cells migrate to lymphaticorgans, particularly T cell rich regions of the lymph nodes, where Tcell activation occurs. Therefore, although administration of theantigen and monocyte-derived dendritic cells may be localized, theresulting immune response is not limited to the tissue ofadministration.

Conditions of particular interest for use with the disclosed methodsinvolve situations where the host response is sub-optimal, for examplein conditions of chronic infection, a lack of immune response to tumorantigens, poor responsiveness to allergens, and the like. In one aspect,the antigen is a tumor antigen and is used to enhance the host's immuneresponse to tumor cells present in the host. In this context, the methodcan be either therapeutic or prophylactic in nature.

Mammalian species that may require enhancement of an immune responseinclude canines and felines; equines, bovines, ovines, porcines, etc.,and primates, particularly humans. Animal models such as primate,canine, or transgenic mouse models can be used for experimentalinvestigations. Animal models of interest include models that involvethe up-regulation of immune responses to tumors, allergens and/orinfection.

A. Methods for Inhibiting or Treating Tumors

Methods are disclosed for inhibiting tumors, for example, inhibitingformation of a tumor, treating a tumor, or reducing the risk ofdeveloping a tumor by delivering 15 kD granulysin to a subject, eitheralone or in combination with one or more other anti-tumor agents. Insome embodiments, methods are disclosed for inhibiting conversion of abenign tumor to a malignant tumor, or inhibiting metastasis. The tumorcan be any tumor, including, but not limited to, tumors of theesophagus, lung, liver, kidney, skin, colon and gastrointestinal tract.In some examples, the tumor can be a mesothelioma, or stomach cancer. Inother examples, the tumor is a skin tumor, such as, but not limited to,a squamous cell carcinoma or a basal cell carcinoma.

The methods disclosed include selecting a subject in need of treatmentfor the condition and administering to the subject a therapeuticallyeffective amount of 15 kD granulysin. Additional agents, such asanti-bacterial, anti-viral, or other therapeutic agents, such as achemotherapeutic agent, can also be administered to the subject.However, in other embodiments substantially pure 15 kD granulysin isadministered, for example 15 kD granulysin in the substantial orcomplete absence of 9 kD granulysin.

In several embodiments, the disclosure is further directed to anti-tumormethods for decreasing the risk of developing a tumor in a subjectexposed to a carcinogen, or inhibiting or delaying the development of atumor. The tumor can be, for example, skin cancer, such as basal cellcarcinoma, keratinocyte carcinoma or squamous cell carcinoma. In anotherexample, the tumor can be an esophageal, stomach, lung, kidney, brain,or colon tumor. In other embodiments, 15 kD granulysin is used for theinhibition of mesothelioma.

Treatment of the conditions described herein can be prophylactic or,alternatively, can be initiated after the development of a conditiondescribed herein. Treatment that is prophylactic, for instance, can beinitiated before a subject manifests symptoms of a condition. In someexamples, such as for skin cancer, treatment can be initiated before orduring exposure to an agent that damages DNA, such as a result of anexposure to a carcinogen, UV light, oxidative stress, alkylation damageor deamination. In some examples, treatment can be following theexposure to the DNA damaging agent, but before the appearance of atumor. In some examples, treatment can occur before or during exposureto a carcinogen, such as an occupational exposure, e.g., asbestos, orsmoking. Treatment prior to the development of the condition is referredto herein as treatment of a subject that is “at risk” of developing thecondition. Accordingly, administration of 15 kD granulysin can beperformed before, during, or after the occurrence of the conditionsdescribed unless otherwise indicated herein.

Treatment initiated after the development of a condition may result indecreasing the severity of the symptoms of one, or more, of theconditions, or completely removing the symptoms, or reducing metastasis.

Non-limiting examples of subjects particularly suited to receiving 15 kDgranulysin before diagnosis of disease include those whose skin may beexposed to excessive natural or artificial UV irradiation, or subjectswho are exposed to a carcinogen due to an occupational exposure, such asan industrial chemical, or due to smoking, or exposure to anon-infectious agent. In one example, a subject who has been exposed toasbestos or silica is at risk for developing mesothelioma. Alternativelythe subject may be someone with a genetic predisposition to develop atumor (such as a family history of cancer, such as breast, lung or coloncancer), an infectious risk factor that predisposes to tumor development(such as HPV exposure or HCV or Epstein-Barr virus infection), or agenetic disorder that predisposes to tumor development (such asGardner's syndrome, xeroderma pigmentosum, Fanconi's anemia, Bloom'ssyndrome, or familial adenomatous polyposis). In particular examples thesubject has an immunodeficiency (such as HIV infection or a drug-inducedimmunodeficiency as in someone who has undergone an organ transplant andis taking immunosuppressive therapy). In another example, the subjecthas an inherited immunodeficiency (such as ataxia telangiectasia orWiskott-Aldrich syndrome).

In some examples, the subject has not yet developed a tumor. In otherexamples, the subject has a benign tumor that can convert into amalignant or even metastatic tumor. For example, the subject may be asmoker who has not developed lung cancer; subjects exposed to large orexcessive amounts of UV light, but who have not developed skin cancer,such as melanoma or basal cell carcinoma; or a subject with a familialdisposition to develop melanoma, for example a mutation in the CDKN2A,KIT, MDM2, or BRAF gene, or a diagnosis of xeroderma pigmentosum,retinoblastoma, Werner syndrome, hereditary breast and ovarian cancer,or Cowden syndrome. In other embodiments a subject is selected who hasclinical risk factors for developing melanoma, such as dysplastic nevi,extensive freckling, or a past history of one or multiple melanomas. Inone aspect of the invention, formation of tumors is delayed, inhibitedor decreased. The types of tumors that may occur in response to an agentthat damages DNA in the skin include actinic keratosis, basal cellcarcinoma, squamous cell carcinoma, and melanoma.

Whether the formation of tumors in a subject is reduced can bedetermined for example, by the use of animal models, for instancetransgenic mice that have been exposed to solar-simulated light orexposure to sunlight, or using a model wherein an animal is exposed to aDNA alkylating agent. Solar-simulated light is light having a spectralprofile which is similar to natural solar irradiation, e.g. the emissionspectrum of a solar simulator looks similar to spectrum of a solar noonday. Wavelengths of light include about 295-400 nm so is inclusive ofUVA and UVB, but not UVC which does not penetrate the ozone layer of theatmosphere (see, for instance, Yoon et al., J. Mol. Biol. 299:681-693,2000). However, the methods are of use with any initiating agent,including agents known to cause cancer (such as the carcinogens intobacco smoke). In some embodiments, the subject is at risk of exposureto an initiating agent due to an occupational exposure.

In another embodiment, the presence of a tumor can be determined bymethods known in the art, and typically include cytological andmorphological evaluation. The cells suspected of being cancerous can bein vivo or ex vivo, including cells obtained from a biopsy.

The composition including 15 kD granulysin may be formulated in avariety of ways for administration to a subject to delay, inhibit,reduce the risk of developing, or treat a tumor of interest. Forexample, the composition can be formulated for application such that itinhibits metastasis of an initial lesion.

The 15 kD granulysin can be administered to slow or inhibit the growthof cells, such as tumor cells, or to inhibit the conversion of a benignlesion to a malignant one. In these applications, a therapeuticallyeffective amount of 15 kD granulysin is administered to a subject in anamount sufficient to inhibit growth, replication or metastasis of tumorcells, or to inhibit an indication or a symptom of the tumor. In someembodiments, 15 kD granulysin is administered to a subject to inhibit orprevent the development of metastasis, or to decrease the number ofmicrometastases, such as micrometastases to the regional lymph nodes(Goto et al., Clin. Cancer Res. 14:3401-3407, 2008).

Pharmaceutical compositions of 15 kD granulysin may also include anadditional therapeutic agent, for example, an anti-inflammatory agent, aco-stimulatory molecule, a TLR agonist (such as LPS), a cytokine (suchas IL-4), a UV protectant or an additional chemotherapeutic agent.Contemplated herein are pharmaceutical compositions in which the 15 kDgranulysin is co-administered with the additional therapeutic agent. Inone embodiment, the 15 kD granulysin may be administered before, after,or during the administration of the additional therapeutic agent. Inanother example, the 15 kD granulysin can be administered up to about 3days prior to the administration of the additional therapeutic agent toprime the immune system, thus providing a robust and sufficient immuneresponse following administration of the additional therapeutic agent totreat, ameliorate or delay the onset of the disease or disorder.

Pharmaceutical compositions for the treatment of at least, but notlimited to, the above conditions are thus provided for both local (suchas topical or inhalational) use and for systemic (such as oral orintravenous) use. Therefore, the disclosure includes within its scopepharmaceutical compositions formulated for use in human or veterinarymedicine. While the composition will typically be used to treat humansubjects it may also be used to treat similar or identical diseases inother vertebrates, such as other primates, dogs, cats, horses, and cows.A suitable administration format may best be determined by a medicalpractitioner for each subject individually. Various pharmaceuticallyacceptable carriers and their formulation are described in standardformulation treatises, e.g., Remington: The Science and Practice ofPharmacy, The University of the Sciences in Philadelphia, Editor,Lippincott, Williams, & Wilkins, Philadelphia, Pa., 21^(st) Edition(2005). See also Wang, Y. J. and Hanson, M. A., Journal of ParenteralScience and Technology, Technical Report No. 10, Supp. 42: 2S, 1988.Generally, the dosage form of the pharmaceutical composition will bedetermined by the mode of administration chosen. Additional informationabout such pharmaceutical compositions is provided in Example 10.

The 15 kD granulysin can be co-administered with a target antigen. Inone embodiment, the antigen may include a DNA vaccine. For example, theDNA vaccine can be a Human Papilloma Virus (HPV) vaccine administeredwith or shortly after (up to about 3 days) administration of the 15 kDgranulysin to treat or control infection by the virus. In anotherembodiment, the target antigen is a tumor antigen such asprostate-specific membrane antigen (PSMA). PSMA expression issignificantly elevated in carcinoma of the prostate, particularly inmetastastic disease and recurrent disease after hormone therapy(androgen deprivation) fails. These properties make PSMA an ideal targetfor anti-cancer vaccines. In one example, 15 kD granulysin can beadministered simultaneously or up to about 3 days after administrationof PMSA to the host. The co-administration of PMSA and 15 kD granulysinpermits “priming” of the immune system via differentiation of monocytesinto monocyte-derived dendritic cells, which in turn enhance antigenpresentation to naïve T cells. In addition, the monocyte-deriveddendritic cells can be stimulated to secrete cytokines, such asinterferon-γ and IL-12 particularly desirable in cancer immunotherapy.

In some embodiments, the compositions disclosed are utilized in a “primeboost” regimen. An example of a “prime boost” regime may be found inYang et al., (J. Virol. 77:799-803, 2002), which is incorporated hereinby reference. In these embodiments, the 15 kD granulysin is delivered toa subject, thereby “priming” the immune response of the subject, andthen a second immunogenic composition such as a DNA vaccine is utilizedas a “boost” vaccination. In one embodiment, a priming composition and aboosting composition are combined into a single formulation. Forexample, a single formulation may comprise 15 kD granulysin as thepriming component and a vector expressing PMSA as the boostingcomponent. In this example, the compositions may be contained in asingle vial where the two components are mixed together. In anotherembodiment, the priming composition may be administered simultaneouslywith the boosting composition, but in separate formulation where thepriming and boosting compositions are separated.

The terms “priming” and “boosting” as used herein may refer to theinitial and subsequent immunizations, respectively, e.g., in accordancewith the definitions these terms normally have in immunology. However,in certain embodiments, e.g., where the priming component and boostingcomponent are in a single formulation, initial and subsequentimmunizations may not be necessary as both the “prime” and the “boost”compositions are administered simultaneously.

In other examples, the 15 kD granulysin is co-administered with a targetantigen in the form of a fusion protein. In some examples, 15 kDgranulysin is administered to a subject as a fusion protein with a tumorantigen, such as a prostate tumor antigen (for example, PMSA orprostatic acid phosphatase (PAP)). Methods for making fusion proteinsare well known to those skilled in the art. For example U.S. Pat. No.6,057,133 to Bauer et al. (herein incorporated by reference) disclosesmethods for making fusion molecules composed of human interleukin-3(hIL-3) variant or mutant proteins functionally joined to a secondcolony stimulating factor, cytokine, lymphokine, interleukin,hematopoietic growth factor or IL-3 variant. Similar methods can be usedto generate fusion proteins including 15 kD granulysin linked to otheramino acid sequences, such as a target antigen (for example, PAP).Linker regions can be used to space the two portions of the protein fromeach other and to provide flexibility between them. The linker region isgenerally a polypeptide of between 1 and 500 amino acids in length, forexample less than 30 amino acids in length, for example between 5 and 20amino acids in length. The linker joining the two molecules can bedesigned to (1) allow the two molecules to fold and act independently ofeach other, (2) not have a propensity for developing an orderedsecondary structure which could interfere with the functional domains ofthe two proteins, (3) have minimal hydrophobic or charged characteristicwhich could interact with the functional protein domains and (4) providesteric separation of the two regions. Typically surface amino acids inflexible protein regions include Gly, Asn and Ser. Other neutral aminoacids, such as Thr and Ala, can also be used in the linker sequence.Additional amino acids can be included in the linker due to the additionof unique restriction sites in the linker sequence to facilitateconstruction of the fusions. Other moieties can also be included, asdesired. These can include a binding region, such as avidin or anepitope, such as a polyhistidine tag, which can be useful forpurification and processing of the fusion protein. In addition,detectable markers can be attached to the fusion protein, so that thetraffic of the fusion protein through a body or cell can be monitoredconveniently. Such markers include radionuclides, enzymes, fluorophores,and the like.

Fusing of a 15 kD granulysin nucleic acid sequence with a nucleic acidsequence encoding another protein can be accomplished by the use ofintermediate vectors. Alternatively, one gene can be cloned directlyinto a vector containing the other gene. Linkers and adapters can beused for joining the nucleic acid sequences, as well as replacing lostsequences, where a restriction site was internal to the region ofinterest. Genetic material (DNA) encoding one polypeptide, peptidelinker, and the other polypeptide is inserted into a suitable expressionvector which is used to transform prokaryotic or eukaryotic cells, forexample bacteria, yeast, insect cells or mammalian cells. Thetransformed organism is grown and the protein isolated by standardtechniques, for example by using a detectable marker such asnickel-chelate affinity chromatography, if a polyhistidine tag is used.The resulting product is therefore a new protein, a fusion protein,which includes 15 kD granulysin joined by a linker region to a secondprotein or a portion of a second protein (such as a target antigen). Toconfirm that the fusion protein is expressed, the purified protein issubjected to electrophoresis in SDS-polyacrylamide gels, and transferredonto nitrocellulose membrane filters using established methods. Theprotein products can be identified by Western blot analysis usingantibodies directed against the individual components, such as apolyhistidine tag and PA.

The nucleic acid sequence encoding a 15 kD granulysin fusion protein canbe under the control of a suitable promoter. Suitable promoters include,but are not limited to, the gene's native promoter, retroviral LTRpromoter, or adenoviral promoters, such as the adenoviral major latepromoter, the CMV promoter, the RSV promoter, inducible promoters, suchas the MMTV promoter, the metallothionein promoter, heat shockpromoters, the albumin promoter, the histone promoter, the α-actinpromoter, TK promoters, B19 parvovirus promoters, and the ApoAIpromoter.

In one embodiment, a composition including 15 kD granulysin and anantigen are delivered to a subject by methods described herein, therebyachieving an effective therapeutic and/or an effective prophylacticimmune response. Additional information about modes of administering 15kD granulysin is provided in Example 11.

The therapeutically effective amount of 15 kD granulysin will bedependent on the subject being treated, the severity and type of thecondition, and the manner of administration. For example, atherapeutically effective amount of 15 kD granulysin can vary from about0.01 μg per kilogram (kg) body weight to about 1 g per kg body weight,such as about 1 μg to about 5 mg per kg body weight, or about 5 μg toabout 1 mg per kg body weight. The exact dose is readily determined byone of ordinary skill in the art based on the potency of the specificformulation, the age, weight, sex and physiological condition of thesubject.

A therapeutically effective amount of 15 kD granulysin can beadministered with an antigen from which a subject requires protection.In one example, the target antigen is a tumor antigen, for example, anoncofetal antigen (e.g., carcinoembryonic antigen (CEA),alpha-fetoprotein or an antigen from the MAGE family). In a specific,non-limiting example, the tumor antigen can be a differentiationantigen, for example, a melanoma differentiation antigen (e.g., MART-1,MAGE 1, MAGE 3, gp 100, or tyrosinase). In another embodiment, thetarget antigen can be a vaccine, such as a DNA vaccine (e.g., HPV, HBVor EBV vaccine). The amount of target antigen administered to thesubject is dependent on a number of factors, such as the condition beingtreated, the severity of the condition, route of administration and theanticipated duration of treatment. It will be apparent to one of skillin the art that the amount or concentration of antigen to beadministered to the subject can be determined by conventional means byan attending physician or veterinarian.

In one embodiment for the inhibition and/or treatment of melanoma, atherapeutically effective amount of 15 kD granulysin can be administeredin conjunction with surgery and/or with another therapeutic agent, suchas a chemotherapeutic agent (e.g., 5-fluorouracil, cisplatin,paclitaxel, doxorubicin or cyclophosphamide). In another specific,non-limiting example, a therapeutically effective amount of 15 kDgranulysin can be used for the treatment of a tumor in conjunction withadministration of a cytokine, for example interleukin-4 (IL-4) and atoll-like receptor agonist, for example, lipopolysaccharide. In thisexample, the cytokine and toll-like receptor agonist can be administeredprior to, during, or after administration of the 15 kD granulysin.

In another embodiment, it is contemplated that a monocyte can be removedfrom a subject, and that the monocyte can be primed and manipulated exvivo to become a dendritic cell. The dendritic cell may be administeredback into the subject, as a method of directly increasing the number ofdendritic cells in the subject. Alternatively, the dendritic cell can bemanipulated ex vivo by exposure to a target antigen. The target antigenwill be processed by the dendritic cell and the antigen-presentingdendritic cell can be re-introduced into the subject, thereby activatingan immune response in the subject if the target antigen is present. Asdescribed herein, the therapeutically effective amount of 15 kDgranulysin can be administered with a therapeutically effective amountof at least one additional therapeutic agent, such as a cytokine, achemokine, a toll-like receptor, a chemotherapeutic agent, ananti-microbial agent, an anti-inflammatory agent (such as a steroidalanti-inflammatory agent or a non-steroidal anti-inflammatory agent) or acombination thereof.

B. Methods for Inducing Dendritic Cell Maturation

Monocytes are produced in the bone marrow from hematopoietic stem cellscalled monoblasts. Monocytes circulate in the blood vessels for aboutone to about three days and then typically move to tissues throughoutthe body. In response to inflammation stimuli, monocytes migrate fromthe blood vessels to the site of infection or inflammation where withother cells and factors the monocytes can initiate an immune response.

According to one embodiment, methods of producing dendritic cells frommonocytes are disclosed. The method includes contacting a monocyte (MO)with a therapeutically effective amount of 15 kD granulysin, therebyinducing differentiation of the monocyte into a monocyte-deriveddendritic cell (MO-DC). In one embodiment, an additional agent thatenhances dendritic cell maturation is administered in conjunction with15 kD granulysin. In another example, an additional dendritic cellmaturation agent may be administered after the monocyte differentiationstep has taken place. Specific, non-limiting examples of additionalagents that enhance dendritic cell maturation include, but are notlimited to, granulocyte macrophage colony stimulating factor (GM-CSF),macrophage colony stimulating factor (M-CSF), flt-3, interleukin-4(IL-4), Toll-Like Receptor (TLR) agonists (such as polyriboinosinicpolyribocytidylic acid (poly (I:C)), lipopolysaccharide (LPS), TumorNecrosis Factor-alpha (TNF-α), CpG motif-containing oligonucleotides,imiquimod, interleukin-6 (IL-6), interleukin-13 (IL-13), interleukin-7(IL-7), interferon-alpha (IFN-α), heparan sulfate, calcium ionophore, ora combination of two or more thereof. In one embodiment, the monocyte iscontacted with a therapeutically effective amount of 15 kD granulysin inthe presence of GM-CSF and IL-4.

GM-CSF as defined herein includes the gene product of the human GM-CSFgene and naturally occurring or engineered variants thereof. Thenucleotide and the amino acid sequence of the human GM-CSF is found inGenbank Accession no. NM_000758 (incorporated herein by reference aspresent in GenBank on Oct. 8, 2010). In addition, some naturallyoccurring variants of GM-CSF are listed in NM_000758. GM-CSF is alsoknown as colony stimulating factor 2 (CSF2) and is anticipated as a formof GM-CSF. The invention also includes the use of derivatives of GM-CSFthat retain the biological activity of wild-type GM-CSF, e.g., that inthe presence of 15 kD granulysin stimulate the differentiation ofmonocytes to monocyte-derived dendritic cells. A derivative of GM-CSFincludes a fragment, fusion or modification or analogue thereof, or afusion or modification of a fragment thereof. A fragment of GM-CSFincludes any portion of the glycoprotein that stimulates the productionof monocyte-derived dendritic cells in the presence of 15 kD granulysin.It is preferred that the fragment has at least 50%, at least 70%, or atleast 90% of the activity of full-length GM-CSF. In another embodiment,the fragment has 100% or more of the activity of full-length GM-CSF.

The invention also contemplates a fusion protein of full-length GM-CSF,or a fragment thereof, to another compound. In one embodiment, thefusion protein retains at least 50%, preferably at least 70%, and morepreferably at least 90% of the activity of full-length GM-CSF. Inanother embodiment, the fusion protein retains at least 100% of theactivity of full-length GM-CSF.

The derivatives as described above may be made using protein chemistrytechniques for example by using partial proteolysis, or de novosynthesis. Alternatively, the derivatives may be made by recombinant DNAtechnology. Suitable techniques for cloning, manipulation, modification,and expression of nucleic acids, and purification of expressed proteins,are well known in the art and are described for example in Sambrook etal (2001) “Molecular Cloning, a Laboratory Manual”, 3^(rd) edition,Sambrook et al (eds), Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., USA, incorporated herein by reference.

The invention also includes modifications of full-length GM-CSF or afragment thereof, that stimulates the production of monocyte-deriveddendritic cells from their progenitor cells and which in the presence of15 kD granulysin cause monocytes to differentiation into dendritic cellsand express dendritic cell phenotype markers such as CD86, CD11c, CD83and HLA-DR.

Modifications of full-length GM-CSF include deglycosylating theglycoprotein either fully or partially. Other modifications include afull-length GM-CSF, or a fragment thereof, having a differentglycosylation pattern from that found in naturally occurring humanGM-CSF. Other modifications of full-length GM-CSF, or a fragmentthereof, include amino acid insertions, deletions and substitutions,either conservative or non-conservative, at one or more positions. Suchmodifications may be called analogues of GM-CSF. As defined herein“conservative substitutions” include combinations such as Gly, Ala; Val,Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. Suchmodifications may be made using the methods of protein engineering andsite-directed mutagenesis, as described in Sambrook et al.

GM-CSF and analogues thereof are described in at least the followingpublications, each of which is incorporated herein by reference to theextent that they describe GM-CSF and its analogues; U.S. Pat. No.5,229,496; U.S. Pat. No. 5,391,485; U.S. Pat. No. 5,393,870; U.S. Pat.No. 5,602,007; Wong et al. (Science 228:810-815, 1985); Lee et al.,(Proc. Natl. Acad. Sci. USA 82:4360-4364, 1985); and Cantrell et al.,(Proc. Natl. Acad. Sci. USA 82:6520-6254, 1985).

While it is preferred that GM-CSF is human GM-CSF, GM-CSF from otherspecies can also be used. However, it is anticipated that forapplication in which GM-CSF is administered to a subject, the GM-CSF ispreferably from the same species as the subject. Thus, if the GM-CSF isto be administered to a human subject, the GM-CSF is preferably humanGM-CSF.

In one embodiment, GM-CSF suitable for the practice of this invention isSargramostim, the proper name for yeast-derived recombinant humanGM-CSF, sold under the trade name Leukine® marketed by Bayer HealthCarePharmaceuticals (Morristown, N.J.). Leukine® is a recombinant humanGM-CSF produced in S. cerevisiae expression system. Leukine® is aglycoprotein of 127 amino acids characterized by 3 primary molecularspecies. The amino acid sequence of Leukine® differs from human GM-CSFby a substitution of leucine at position 23, and the carbohydrate moietymay be different from the native protein.

Typically, to generate dendritic cells in vitro, it is useful to purifymonocytes and monocyte precursors from other contaminating cell types.This is commonly achieved through adherence of monocytes to a plasticpolystyrene surface, since monocytes have a greater tendency to adhereto plastic than other cell types found in peripheral blood. Aftersubstantially removing contaminant cells, for example by vigorouswashing, the monocytes can be cultured with agents, such as cytokinesthat differentiate monocytes into dendritic cells. For example, Sallustoand Lanzavecchia (J. Exp. Med. 179:1109-1118, 1994) disclose a methodfor differentiating a monocyte precursor into an immature dendriticcell.

It has been previously reported that the 9 kD form of granulysinactivates monocytes to produce chemokines, including MCP-1 and RANTES,and cytokines, such as IFN-γ (Krensky Biochem. Pharmacol. 59:317-320,2000). One of skill in the art can readily identify without undueexperimentation the concentration of cytokines required for use with 15kD granulysin. In one specific, non-limiting example, cytokines arepresent in a concentration of about 10 ng/ml to about 100 ng/ml,depending on the specific cytokine or cytokine cocktail to be used.Without being bound by theory, it is believed that agents such as GM-CSFand/or IL-4 act synergistically with 15 kD granulysin to enhancedendritic cell maturation.

In one embodiment, a monocyte is contacted with 15 kD granulysin invitro to differentiate the monocyte into a monocyte-derived dendriticcell. To increase the number of monocyte cells in an animal, includinghumans, the subject can be treated with substances which stimulatehematopoiesis, such as GM-CSF or a CpG motif-containing oligonucleotide.For example, U.S. Pat. No. 5,994,126 discloses methods for isolatingdendritic cell precursors and methods for increasing the number ofdendritic cell precursors in a sample. Additionally, Krug et al. (J.Immunol. 170:3468-3477, 2003) disclose methods for producing andisolating monocyte-derived dendritic cells in culture upon incubationwith CpG motif-containing oligonucleotides.

Thus, a monocyte can be contacted with a therapeutically effectiveamount of 15 kD granulysin for a sufficient period of time todifferentiate into a dendritic cell in vitro. In one specific,non-limiting example, peripheral blood mononuclear cell cultures (PBMCs)are contacted with a therapeutically effective amount of 15 kDgranulysin for a sufficient period of time to differentiate into maturedendritic cells in vitro. In one example, a culture of isolatedperipheral blood monocytes containing about 1 to about 4×10⁶ cells/mlare treated with a therapeutically effective amount of 15 kD granulysinin vitro. In this example, the concentration of 15 kD granulysineffective to induce maturation of the monocytes is about 1 nM to about20 nM, or is about 5 nM to about 15 nM, or is about 10 nM. In oneembodiment, the culture is maintained for at least one day. In anotherembodiment, the culture is maintained for about 2 days to about 14 days.In another embodiment, the culture is maintained for about 3 days toabout 6 days.

A method for inducing differentiation of monocytes in vitro in thepresence of a target antigen is disclosed. The method includescontacting a monocyte with a therapeutically effective amount of 15 kDgranulysin and a therapeutically effective amount of a target antigen,thereby differentiating the monocyte into a monocyte-derived dendriticcell expressing the antigen of interest in vitro. In this instance, themonocyte can be contacted with the antigen of interest sequentially orsimultaneously. The antigen can be any antigen, including, but notlimited to, a tumor antigen, an antigen from a non-infectious agent, anallergen, or an antigen of use in a vaccine. Thus, in one embodiment, amonocyte is contacted with a therapeutically effective amount of 15 kDgranulysin to produce a dendritic cell. In other examples, a monocyte iscontacted with a therapeutically effective amount of 15 kD granulysinfused to an antigen (such as a tumor antigen) to produce a dendriticcell.

In a further embodiment, the dendritic cell is contacted with atherapeutically effective amount of a target antigen to inducepresentation of the antigen by the dendritic cell. Thus, a matureantigen-presenting cell (APC) is produced by this method.

Exemplary antigens include, but are not limited to, epitopes or antigensfrom tumors, non-infectious agents or allergens. These antigens may becomposed of protein, DNA, RNA, lipid, sugar, whole cell lysates,apoptotic cells, or any combination thereof. Some preferred antigensinclude soluble tumor protein antigens (such as CEA, MAGE-1, MART 1,tyrosinase), tumor-derived RNA, unfractionated acid-eluted peptides fromthe MHC class I molecules of tumor cells, and recombinant, purified, orinactivated vaccine proteins. Antigens of interest further includepolypeptides and other immunogenic biomolecules, which can be producedby recombinant methods or isolated from natural sources. Complexantigens such as cell lysates inactivated (e.g. heat killed) viruses,bacterial cells or fractions thereof are also of use.

Potential tumor antigens for immunotherapy include, but are not limitedto, tumor specific antigens, e.g. immunoglobulin idiotypes and T cellantigen receptors; oncogenes, such as p21/ras, p53, p210/bcr-abl fusionproduct; developmental antigens, e.g. MART-1/Melan A, MAGE-1, MAGE-3;GAGE family; telomerase; viral antigens, e.g. human papilloma virus;Epstein Barr virus; Hepatitis B virus; tissue specific self-antigens,e.g. tyrosinase, gp100, prostatic acid phosphatase, prostate specificantigen, PSMA, thyroglobulin, α-fetoprotein; and over-expressedself-antigens, e.g. her-2/neu; carcinoembryonic antigen, muc-1, and thelike. Tumor cell derived protein extracts or RNA may be used as a sourceof antigen, in order to provide multiple antigens and increase theprobability of inducing immunity to more than one tumor associatedantigen.

As an alternative to injecting the target antigen into the host,endogenous tissue samples expressing the antigen can be used as anendogenous source of the antigen. For example, tumor cells that expressa tumor antigen maybe injected alone, or in combination with a dendriticcell maturation agent, to serve as the source of tumor antigen.Administration of an endogenous tumor antigen and dendritic cellmaturation agent in vivo leads to the activation and differentiation ofmonocytes that migrate to the tumor site and can process the endogenoustumor antigen. The processed tumor antigen is expressed on the surfaceof monocyte-derived dendritic cells that can interact with naïve T cellsto produce an immune response against the target antigen.

It is to be appreciated that to induce tolerance to an antigen, aderivative of the antigen may be administered to the subject, and notthe antigen itself. By derivative of an antigen it includes any portionof the antigen which can be presented by a class I or class II MHCmolecule, and which induces tolerance to the antigen itself.

When the antigen is a protein, a derivative of the antigen is typicallya peptide fragment of the antigen including a contiguous sequence ofamino acids of the antigen capable of MHC binding. In one embodiment,the antigen is a fragment between about 6 and about 100 amino acids inlength. In another embodiment, the antigen is a fragment between about 6and about 50 amino acids in length. In yet another embodiment, theantigen is a fragment that is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.

A derivative of the antigen may include a fusion of the antigen, or afusion of a fragment of the antigen to another compound, and which canbe recognized by either a class I or class II MHC molecule whenpresented on the monocyte-derived dendritic cell. Unless the contextindicates otherwise, wherever the term “antigen” is used in the contextof an antigen, a derivative as herein defined is included.

The antigen can be delivered to the monocyte-derived dendritic cell viaany method known in the art, including, but not limited to, pulsing thecells directly with the antigen, or utilizing a broad variety of antigendelivery vehicles, such as, for example, liposomes, or other vectorsknown to deliver antigen to cells. In one specific, non-limiting examplean antigenic formulation includes about 0.1 μg to about 100 mg, or about10 μg to about 10 mg of a selected antigen. An antigen preparation canalso contain buffers, excipients, and preservatives, amongst otheringredients.

In another embodiment, a monocyte is contacted with 15 kD granulysin andan antigen to produce an antigen-presenting differentiated dendriticcell. The cells are contacted with antigen for a time sufficient toallow the antigen to be internalized, processed, and presented by themonocyte-derived dendritic cell. Accordingly, the present invention alsorelates to methods for generating enriched populations of mature,antigen-presenting dendritic cells that can function to present antigento T cells. In one specific non-limiting example, a monocyte iscontacted with 15 kD granulysin and an antigen simultaneously. In oneexample, the monocyte is contacted with a fusion protein including 15 kDgranulysin and an antigen. In another embodiment, the monocyte iscontacted with a composition including 15 kD granulysin to produce amonocyte-derived dendritic cell, which is subsequently or simultaneouslycontacted with an antigen to generate a mature antigen presentingdendritic cell.

In one specific, non-limiting example, monocyte-derived dendritic cellsare obtained in vitro by culturing monocytes with 15 kD granulysin forabout 24 to about 48 hrs. In another specific, non-limiting example,antigen-presenting monocyte-derived dendritic cells are obtained invitro by culturing monocytes with 15 kD granulysin for about 48 to about96 hours, and then contacting the monocyte-derived dendritic cells withan antigen for a time sufficient to allow the antigen to beinternalized, processed, and presented by the monocyte-derived dendriticcell, thereby producing antigen presenting monocyte-derived dendriticcells. In another example, antigen-presenting monocyte-derived dendriticcells are obtained in vitro by culturing monocytes with a fusion proteinincluding 15 kD granulysin and a target antigen for about 24 to about 72hours. In another aspect, the monocytes are contacted with 15 kDgranulysin in vivo. In a further embodiment the antigen presentingmonocyte-derived dendritic cells are incubated with a dendritic cellmaturation agent (e.g., LPS) thereby producing mature antigen-presentingdendritic cells.

One of skill in the art can readily identify monocyte-derived dendriticcells and antigen presenting dendritic cells. These techniques include,but are not limited to, analysis of cell morphology, detection ofspecific antigens present on the cell surface of mature dendritic cellswith for example, monoclonal antibodies, or assays for mixed lymphocytereactions.

In one embodiment, the presence of mature dendritic cells can beconfirmed by antibodies specific for various mature dendritic cellsurface markers, such as CD83, CD40, CD86 and HLA-DR. Typically, labeledantibodies specifically directed to the marker are used to identify thecell population. The antibodies can be conjugated to other compoundsincluding, but not limited to, enzymes, magnetic beads, colloidalmagnetic beads, haptens, fluorochromes, metal compounds, radioactivecompounds or drugs. The enzymes that can be conjugated to the antibodiesinclude, but are not limited to, alkaline phosphatase, peroxidase,urease and β-galactosidase. The fluorochromes that can be conjugated tothe antibodies include, but are not limited to, fluoresceinisothiocyanate, tetramethylrhodamine isothiocyanate, phycoerythrin,allophycocyanins and Texas Red. For additional fluorochromes that can beconjugated to antibodies see Haugland, R. P., Molecular Probes: Handbookof Fluorescent Probes and Research Chemicals (1992-1994). The metalcompounds that can be conjugated to the antibodies include, but are notlimited to, ferritin, colloidal gold, and particularly, colloidalsuperparamagnetic beads.

Mature dendritic cells may also be identified histologically, byassessing nuclear re-organization, vacuole formation, cytoplasmicenlargement, and membrane ruffling. In addition, one of skill in the artcan assess typical mature dendritic cell morphology, including stellateshape and/or well defined veils. For example, one of ordinary skill inthe art would associate the conversion of a monocyte into a dendriticcell with an increase in total cell size (volume) and an increase incell granularity, as can be determined by example by flow cytometry orFluorescence-Activated Cell Sorting (FACS).

Compositions including mature antigen-presenting dendritic cells may beused as vaccines adjuvants to elicit or boost immune responses againstantigens. For example, activated, antigen-presenting monocyte-deriveddendritic cells can be used as vaccines to inhibit or prevent futureinfection, or may be used to activate the immune system to treat ongoingdiseases, such as cancer. As disclosed herein, it is believed that theexpression level of 15 kD granulysin can be used as a correlate ofeffective immunity in monitoring vaccines and/or that analogs of 15 kDgranulysin can be used as therapeutic agents.

Accordingly, the compositions disclosed herein are useful when used inconjunction with vaccines such as, but not limited to, those fortreating chronic bacterial infections, e.g. tuberculosis or chronicviral infections such as those associated with herpes simplex 1 virus,herpes simplex 2 virus, human herpes virus 6, measles virus, rubellavirus, human immunodeficiency virus (HIV), human T cell leukemia virusI, human T cell leukemia virus II, varicella-zoster virus, hepatitis Bvirus, hepatitis C virus, hepatitis D virus, human papilloma virus,parvovirus B19, polyomavirus BK, polyomavirus JC, lentivirus,adenovirus, cytomegalovirus, Epstein-Barr virus, and retrovirus.

Specifically, a method of enhancing vaccine efficacy is disclosed. Themethod includes administering to a subject in need thereof, atherapeutically effective amount of a vaccine and a monocyte-deriveddendritic cell produced by contacting a monocyte with an effectiveamount of 15 kD granulysin. In other aspect, the method furthercomprises contacting the monocyte with an agent that enhances dendriticcell maturation. In a preferred embodiment, the 15 kD granulysin issubstantially free of 9 kD granulysin. In another embodiment, the 15 kDgranulysin consists essentially of 15 kD granulysin.

Mature dendritic cells produced by the methods disclosed can also beutilized to produce activated T lymphocytes. The methods disclosedinclude contacting a monocyte-derived dendritic cell with a T lymphocytein vitro, thereby producing an activated T lymphocyte. Mature dendriticcells generated by the methods can be administered to a subject. Maturedendritic cells generated by contacting a monocyte with a compositionincluding 15 kD granulysin in vitro can be administered to a subject topreferentially stimulate immune responses which block allergic responses(e.g. interferon production). Thus, the mature dendritic cells generatedby 15 kD granulysin treatment may be administered to a subject fortreating an allergic condition in that individual. The treatment ofallergic conditions is based on the discovery that 15 kD granulysin maystimulate dendritic cells to produce anti-allergic agents, such asIFN-α, which in turn increased the production of IFN-γ by natural killer(NK) cells and T cells.

The mature dendritic cells generated by the methods described can beused for immunotherapy. In one embodiment, the dendritic cells generatedby the methods can also be used for tumor immunotherapy. In oneembodiment, the mature dendritic cells present a tumor antigen. Thesedendritic cells can be administered to a subject who has a tumor thatexpresses the tumor antigen. In another embodiment, the mature dendriticcells expressing a target antigen are administered in conjunction with achemotherapeutic agent.

In another embodiment, mature dendritic cells produced by contacting amonocyte with 15 kD granulysin are administered to boost an immuneresponse against another antigen. The granulysin and antigen can beadministered together, or sequentially but sufficiently close togetherfor the granulysin to enhance an immune response against the antigen,for example to enhance an allospecific T cell response against theantigen. In one specific, non-limiting example, the antigen is from aninfectious agent, including but not limited to, an antigen from abacterium, virus, parasite or fungus. The dendritic cells can be fromthe same subject (autologous) or can be from a different individual(heterologous).

A method is also disclosed for inducing the differentiation of monocytesin vivo. The method includes administering a therapeutically effectiveamount of 15 kD granulysin to a subject, thereby inducingdifferentiation of monocytes into differentiated dendritic cells in thesubject. The subject can be a mammal, such as a primate. In onespecific, non-limiting example, the subject is a human, howeverveterinary use is contemplated.

As discussed above, in one embodiment, an agent that enhances dendriticcell maturation is administered in conjunction with 15 kD granulysin.Specific, non-limiting examples of agents that enhance dendritic cellmaturation include IL-4 and GM-CSF. In another embodiment, atherapeutically effective amount of 15 kD granulysin is administered inthe absence of an agent that enhances dendritic cell maturation. In yetanother embodiment, a therapeutically effective amount of 15 kDgranulysin is administered to a subject in conjunction with atherapeutically effective amount of an antigen to produce anantigen-presenting differentiated dendritic cell in the subject. In afurther embodiment, an antigen can be co-administered with 15 kDgranulysin, for example, in a liposome, to trigger antigen uptake andmaturation of dendritic cells in vivo and enhance antigen presentationby the dendritic cells to T cells in vivo. Thus, antigen presentationand immunity can be significantly enhanced using the methods describedherein. For example, 15 kD granulysin and a target antigen may beco-administered in solution, or in a delivery vehicle, such as aliposome, which would facilitate delivery and uptake of 15 kD granulysinand antigen by the subject's monocytes.

In another embodiment, compositions including 15 kD granulysin andmonocytes may be used to treat a subject having cancer. As discussedabove, cancer treatments may be based upon the development of anti-tumorvaccines including 15 kD granulysin and a tumor antigen, or 15 kDgranulysin and mature tumor antigen-presenting dendritic cells. Withoutbeing bound by theory, such vaccines not only elicit anti-tumor antibodyproduction, but also activate natural killer cell lytic activity andantibody dependent cellular cytotoxicity (ADCC). Thus, in the lattercase, administration of compositions including 15 kD granulysin and atarget antigen stimulate production of tumor specific cytotoxic immunecells in vivo which actively target and kill the cancer cells.

In a further embodiment, compositions including activated T cells can beproduced in vitro by, for example, co-culturing the matureantigen-presenting dendritic cells prepared according to the invention,with T cells in vitro. Such compositions are useful in adoptiveimmunotherapy, such as for the production of antigen-specific cytotoxicT lymphocytes or for generating antigen-specific T helper cells.

As disclosed herein, 15 kD granulysin can be used to generate maturedendritic cells in vivo. Thus, in one embodiment, a therapeuticallyeffective amount of 15 kD granulysin is administered locally, such as toa specific site in a subject in order to activate and expand monocytesat that site. In another embodiment, a therapeutically effective amountof 15 kD granulysin is administered systemically, such as byintravenous, intramuscular, intradermal, intraarterial, parenteral, orsubcutaneous injection, or by oral administration or inhalation, toinduce differentiation of monocytes into monocyte-derived dendriticcells.

In one embodiment, 15 kD granulysin is administered in a deliverycomplex. The delivery complex can include 15 kD granulysin and atargeting means. Any suitable targeting means can be used. For example,15 kD granulysin can be associated with (e.g., ionically or covalentlybound to, or encapsulated within) a targeting means (e.g., a moleculethat results in higher affinity binding to a target cell, such as a Bcell). A variety of coupling or cross-linking agents can be used to formthe delivery complex, such as protein A, carbodiimide, andN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP). Examples of adelivery complex include a composition including 15 kD granulysinassociated with a sterol (e.g., cholesterol), a lipid (e.g., a cationiclipid, virosome or liposome), and a target cell specific binding agent(e.g., a ligand recognized by target cell specific receptor). Withoutbeing bound by theory, the complex is sufficiently stable in vivo toinhibit or prevent significant uncoupling prior to delivery to thetarget cell, such as a tumor cell. In one embodiment, the deliverycomplex is cleavable such that 15 kD granulysin is released in afunctional form at the target cell.

In another embodiment, 15 kD granulysin is administered in conjunctionwith a pharmacologically acceptable carrier. Pharmacologicallyacceptable carriers (e.g., physiologically or pharmaceuticallyacceptable carriers) are well known in the art. A suitablepharmacological composition can be formulated to facilitate the use of15 kD granulysin in vivo and/or ex vivo. Such a composition can besuitable for delivery of the active ingredient to any suitable host,such as a patient for medical application, and can be manufactured in amanner that is itself known, e.g., by means of conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmacological compositions for use can be formulated in a conventionalmanner using one or more pharmacologically (e.g., physiologically orpharmaceutically) acceptable carriers including excipients, as well asoptional auxiliaries that facilitate processing of the active compoundsinto preparations which can be used pharmaceutically. Proper formulationis dependent upon the route of administration chosen, and whether usewill be an in vivo or an ex vivo use. For use in vivo, administrationcan be either systemic or local. In addition, one of skill in the artcan readily select a suitable route of administration, including, butnot limited to intravenous, intramuscular, intraperitoneal,transmucosal, subcutaneous, transdermal, transnasal, inhalation, andoral administration.

C. Organ Transplantation Rejection

Despite significant advances in understanding of tissue typing andimmunosuppression and the availability of better immunosuppressiveagents, acute rejection remains a serious clinical problem in organtransplantation. In the absence of successful therapies, organ rejectionleads to graft failure in some patients, for example, requiringreinstitution of dialysis and the search for another donor kidney forrenal transplant recipients.

The instant disclosure provides a method for detecting and/or monitoringorgan transplantation rejection in a transplant recipient includingmonitoring the level of 15 kD granulysin in a sample obtained from thetransplant recipient, wherein a significant increase in the level of 15kD granulysin in the sample obtained from the transplant recipient (forexample, as compared to an earlier or pre-transplantation sample) isassociated with organ transplantation rejection.

Also disclosed is a method for inducing tolerance or inhibitingrejection of a cell, tissue, or organ transplant in a transplantrecipient including administering to a mammalian transplant recipient amonocyte-derived dendritic cell produced by exposing a monocyte to acomposition including 15 kD granulysin and a pharmacological agent,wherein the pharmacological agent is capable of inducing immunologicaltolerance in the monocyte-derived dendritic cell, and administering tothe transplant recipient the immunologically tolerant monocyte-deriveddendritic cell, thereby inhibiting rejection of the transplanted cell,tissue or organ. Examples of the types of pharmacological agents thatare capable of inducing immunological tolerance in a monocyte-deriveddendritic cell include, but is not limited to, cytokines (such asGM-CSF, G-CSF, M-CSF, IL10/TGF-β, IFN-γ, TNF-α, Hepatocyte Growth Factor(HGF), IL-16/TPO, IL-21, IL-10, or Thymic Stromal Lymphopoietin (TSLP)),neuropeptides (such as Vasoactive Intestinal Peptide (VIP), Vitamin DReceptors (VDR) agonists, and Toll-Like Receptor (TLR) agonists (such asLPS).

GM-CSF is known to induce murine semi-mature IL-12 dendritic cells withhigh expression of MHC class II and co-stimulatory molecules (Gangi etal., J. Immunol. 174:7006-7013, 2005). VIP is a neuropeptide released byimmune cells in response to antigen stimulation and a potentanti-inflammatory agent. M-CSF and IL-4 can induce monocyte-derivedIL10⁺, IL12^(neg) tolerogenic dendritic cells (Li et al., J. Immunol.174:4706-4717, 2005). G-CSF indirectly favors the in vitrodifferentiation of peripheral blood monocytes to tolerogenic dendriticcells through the release of IL-10 and IFN-α (Rutella et al., Eur. J.Immunol. 34:1291-1302, 2004). HGF is known to skew monocytedifferentiation toward IL-10 producing, co-stimulating tolerogenicdendritic cells (Rutella et al., Blood 108:1435-1440, 2006). TSLP isproduced by epithelial cells of thymic Hassall's corpuscles.TSLP-released thymic dendritic cells express CD80/CD86 and MHC class IIand promote the conversion of thymocytes into regulatory T (Treg) cells(Watanabe et al., Nature 436:1181-1185, 2005).

Other examples of pharmacological agents that are capable of inducingimmunological tolerance in monocyte-derived dendritic cells can befound, for example in Rutella et al., (Blood 108:1435-1440, 2006) andSilk and Fairchild (Curr. Opin. Organ. Transpl. 14:344-350, 2009). Inone example, the methods further comprise administering to the mammaliantransplant recipient at least one immunosuppressant or anti-inflammatorydrug.

D. Autoimmune Disorders

Dendritic cells regulate immune responses that result in two oppositeoutcomes: immunity or tolerance. The fine regulation of these twodistinct functions is not completely understood. However, it ispresently believed that the net effect of antigen dose, dendritic celllineage and maturation status, and dendritic cell stimulation bypathogen derived products, and cytokine milieu at sites of inflammationdetermine whether an immunogenic or a tolerogenic T cell response willdevelop. Because dendritic cell-based immunotherapy in autoimmunedisease depends on tolerogenic dendritic cells, discerning markers fortolerogenic dendritic cells is a significant objective. For example,immature dendritic cells and IL-10-modified dendritic cells have beenobserved to mediate immune tolerance by inducing T-cell anergy orT-helper type 2 responses. A variety of pharmacological agents are knownto induce tolerogenicity in dendritic cells, in some examples, theinduction is attributed to the activity of individual cytokines orneuropeptides. Examples of the types of pharmacological agents that arecapable of inducing immunological tolerance in a monocyte-deriveddendritic cell include GM-CSF, G-CSF, M-CSF, IL10/TGF-β, IFNγ, TNFα,HGF, IL-16/TPO, IL-21, IL-10, TSLP, VIP, VDR agonists, and TLRs.

Autoimmune diseases comprise a large number of widely varying illnesses.Their common feature is the existence of an immune response in thesubject against one or more “self” antigens, including such wide rangingmolecules as proteins, DNA and carbohydrates. These diseases can causesymptoms ranging from mild discomfort to debilitation and death. Most ofthe autoimmune diseases remain enigmatic, not only in their molecularbasis, but in their prediction, progression and treatment. Autoimmunediseases include systemic lupus erythematosus, Sjogren's syndrome,rheumatoid arthritis, juvenile onset diabetes mellitus, Wegener'sgranulomatosis, inflammatory bowel disease, polymyositis,dermatomyositis, multiple endocrine failure, Schmidt's syndrome,autoimmune uveitis, Addison's disease, adrenalitis, Graves' disease,thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease,pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis,atherosclerosis, presenile dementia, demyelinating diseases, multiplesclerosis, subacute cutaneous lupus erythematosus, hypoparathyroidism,Dressler's syndrome, myasthenia gravis, autoimmune thrombocytopenia,idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigusvulgaris, pemphigus, dermatitis herpetiformis, alopecia arcata,pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome(calcinosis, Raynaud's phenomenon, esophageal dysmotility,sclerodactyly, and telangiectasia), adult onset diabetes mellitus (TypeII diabetes), male and female autoimmune infertility, ankylosingspondylitis, ulcerative colitis, Crohn's disease, mixed connectivetissue disease, polyarteritis nedosa, systemic necrotizing vasculitis,juvenile onset rheumatoid arthritis, glomerulonephritis, atopicdermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease,sarcoidosis, rheumatic fever, asthma, recurrent abortion,anti-phospholipid syndrome, farmer's lung, erythema multiforme, postcardiotomy syndrome, Cushing's syndrome, autoimmune chronic activehepatitis, bird-fancier's lung, allergic disease, allergicencephalomyelitis, toxic epidermal necrolysis, alopecia, Alport'ssyndrome, alveolitis, allergic alveolitis, fibrosing alveolitis,interstitial lung disease, erythema nodosum, pyoderma gangrenosum,transfusion reaction, leprosy, malaria, leishmaniasis, trypanosomiasis,Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis,schistosomiasis, giant cell arteritis, ascariasis, aspergillosis,Sampter's syndrome, eczema, lymphomatoid granulomatosis, Behcet'sdisease, Caplan's syndrome, Kawasaki's disease, dengue,encephalomyelitis, endocarditis, endomyocardial fibrosis,endophthalmitis, erythema elevatum et diutinum, psoriasis,erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome,Felty's syndrome, filariasis, cyclitis, chronic cyclitis, heterochroniccyclitis, Fuch's cyclitis, IgA nephropathy, Henoch-Schonlein purpura,glomerulonephritis, graft versus host disease, transplantationrejection, human immunodeficiency virus infection, echovirus infection,cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virusinfection, post vaccination syndromes, congenital rubella infection,Hodgkin's and Non-Hodgkin's lymphoma, renal cell carcinoma, multiplemyeloma, Eaton-Lambert syndrome, relapsing polychondritis, malignantmelanoma, cryoglobulinemia, Waldenstrom's macroglobulemia, Epstein-Barrvirus infection, rubulavirus, and Evan's syndrome.

The present invention provides a method for treating the symptoms of anautoimmune disorder. Preferably, the treatment occurs during thepresymptomatic or preclinical stage of the autoimmune disorder, and insome cases during the symptomatic stage of the disorder. Early treatmentis preferable, in order to reduce, inhibit, or prevent the loss offunction associated with inflammatory tissue damage. The presymptomatic,or preclinical stage will be defined as that period not later than whenthere is T cell involvement at the site of disorder, e.g. islets ofLangerhans, synovial tissue, thyroid gland, but the loss of function isnot yet severe enough to produce the clinical symptoms indicative ofovert disease. T cell involvement may be evidenced by the presence ofelevated numbers of T cells at the site of the disorder, the presence ofT cells specific for auto-antigens, the release of perforins andgranzymes at the site of the disorder, or as a response toimmunosuppressive therapy.

For example, degenerative joint diseases can be inflammatory, as withseronegative spondylarthropathies, e.g. ankylosing spondylitis andreactive arthritis; rheumatoid arthritis; gout; and systemic lupuserythematosus. The degenerative joint diseases have a common feature, inthat the cartilage of the joint is eroded, eventually exposing the bonesurface. Destruction of cartilage begins with the degradation ofproteoglycan, mediated by enzymes such as stromelysin and collagenase,resulting in the loss of the ability to resist compressive stress.Alterations in the expression of adhesion molecules, such as CD44,ICAM-1, and extracellular matrix protein, such as fibronectin andtenascin, follow. Eventually fibrous collagens are attacked bymetalloproteases, leading to the loss of collagenous microskeleton. Atthis point, repair by regeneration is no longer possible. There issignificant immunological activity within the synovium during the courseof inflammatory arthritis. While treatment during early stages isdesirable, the adverse symptoms of the autoimmune disorder may be atleast partially alleviated by treatment, such as the administration of15 kD granulysin, during later stages. Clinical indices for the severityof arthritis include pain, swelling, fatigue and morning stiffness, andmay be quantitatively monitored by Pannus criteria. Autoimmune diseaseprogression in animal models can be followed by measurement of affectedjoint inflammation.

The present invention provides a composition and method of treating orinhibiting an immune-based disease in vivo. In one embodiment, a methodfor treating or ameliorating an autoimmune disease in a subject includesadministering a therapeutically effective amount of 15 kD granulysin invivo to induce differentiation of monocytes in the subject intomonocyte-derived dendritic cells, transforming the monocyte-deriveddendritic cells to tolerogenic dendritic cells by exposure in vivo tofactors that promote tolerogenicity, thereby treating the ongoingautoimmune disease and/or inhibiting its future exacerbation. Forexample, factors that promote tolerogenicity include, but are notlimited to, neuropeptides (such as vasoactive intestinal peptide andpituitary adenylate cyclase-activating polypeptide) (Silk and Fairchild,Curr. Opin. Organ Transplant. 14:344-350, 2009), cytokines (such asG-CSF, IL-4, GM-CSF and HGF) (Rutella et al., Blood 108:1435-1140,2006), toll-like receptors (such as LPS), Tryptophan (Trp) metabolites(Brown et al., 1991), and Vitamin D Receptor (VDR) agonists (Adorini andPenna, Hum. Immunol. 70:345-352, 2009).

In another embodiment, a method for treating or ameliorating anautoimmune disease in a subject includes removing a monocyte from asubject with an autoimmune disease, and treating the monocyte with 15 kDgranulysin to induce differentiation of the monocyte to amonocyte-derived dendritic cell, transforming the monocyte-deriveddendritic cell to a tolerogenic dendritic cell by exposure in vitro tofactors that promote tolerogenicity, and re-introducing the pre-treateddendritic cell to the subject in order to treat the ongoing autoimmunedisease and/or inhibit its future exacerbation. In one example, themethods further include administration of at least one immunosuppressantor anti-inflammatory drug to the subject with the autoimmune disease.

In another embodiment, a method of treating the symptoms of anautoimmune disease include contacting a monocyte with 15 kD granulysinand a target antigen associated with the autoimmune disease in vitro toproduce a monocyte-derived dendritic cell expressing the target antigen,transforming the monocyte-derived dendritic cell expressing the targetantigen to a tolerogenic dendritic cell expressing the target antigen byexposure in vitro to factors that promote tolerogenicity, andre-introducing the pre-treated dendritic cell expressing the targetantigen to the subject in order to treat the symptoms of the autoimmunedisease. In some instances, the antigen of interest is a tumor antigen,such as a lung, colon or breast cancer antigen. In another embodiment,the target antigen can be a vaccine, such as a DNA vaccine (e.g., HBV,HPV, or EBV vaccine). In a further embodiment, the antigen of interestis an autoimmune disease antigen, such as an arthritis-induced antigenor a lupus-associated antigen. In another embodiment, the methodsfurther include administering at least one immunosuppressant oranti-inflammatory drug to the subject with the autoimmune disease. In aspecific, non-limiting example, the autoimmune disease is any one of theconditions described herein.

The disclosure is further illustrated by the following non-limitingExamples.

EXAMPLES Example 1 Differentiation of Monocytes

Granulysin 15 kD was found in cell culture studies to inducedifferentiation of monocytes into monocyte-derived dendritic cells. Theresults are shown in FIG. 1. CD14+ monocyte cells were isolated fromhuman buffy coats using magnetic beads. Cells were plated at 2×10⁶/ml inthe presence of 15 kD granulysin (10 nM), 9 kD granulysin (10 nM) orcell culture medium for 48 hours. The cells were then analyzed forforward and side scatter by Fluorescence-Activated Cell Sorting (FACS).FIG. 1C shows the effect of treatment in the presence of 15 kDgranulysin (10 nM). As noted in FIG. 1C an increase in cell size(x-axis) and an increase in cell granularity (y-axis) was observed, aswould be expected following differentiation of monocytes intomonocyte-derived dendritic cells. In contrast, the treatment with 9 kDgranulysin did not result in an increase in cell size or cellgranularity and was comparable to treatment with cell culture mediumonly, and therefore suggests that 9 kD granulysin does not activatemonocytes in vitro.

Example 2 Cell Surface Expression of Activated Monocytes

Dendritic cells can be distinguished from monocytes by their cellsurface expression of several markers, for example CD40, CD80 and CD83(Chapuis et al., Eur. J. Immunol. 27:431-441, 1997; Zhou and Tedder, J.Immunol. 154:3821-3835, 1995). Monocyte cells were prepared and isolatedas described in Example 1, and were subjected to antibody staining forthe following cell surface markers: CD40, CD80, CD83, and CD209. Thecells were then analyzed by FACS; the results of which are presented inFIG. 2.

FIG. 2A discloses the levels of cell surface marker CD40 after culturingwith 9 kD granulysin (10 nM) or 15 kD granulysin (10 nM) as compared tocell culture medium (control). Monocytes treated with 15 kD granulysindisplayed a significant increase in fluorescence, based on expression ofCD40 as compared to 9 kD treated monocytes or the control sample. Anincrease in fluorescence based expression of cell surface markers CD83and CD80 was also observed when the monocytes were cultured with 15 kDgranulysin for 48 hours prior to FACS analysis. In contrast, monocytestreated with 9 kD granulysin (10 nM) for an equivalent amount of timeshowed fluorescence profiles similar to the control sample (cell culturemedium only). Fluorescence of monocytes treated with 15 kD granulysin(10 nM) as measured by CD209 cell surface expression was also observedto be slightly elevated when compared to monocytes treated with 9 kDgranulysin (under identical conditions). Both CD40 and CD80 areco-stimulatory molecules that participate in the process of presentingantigens to T cells; CD83 is a cell surface marker associated with thecell surface of dendritic cells. Thus, the shift in fluorescence asmeasured by these cell surface markers is indicative of thedifferentiation of monocytes to monocyte-derived dendritic cells and theability of the monocyte-derived dendritic cells to process and presentantigens.

Example 3 Additional Method for Differentiation of Monocytes intoDendritic Cells

As described in Example 2, dendritic cells can be distinguished frommonocytes by the expression of several cell surface markers, for exampleCD83, CD40 and CD80. In this example, cell culture studies demonstratedthat treatment of human CD14+ monocytes with 15 kD granulysin (10 nM)and IL-4 (10 ng/ml) can expeditiously differentiate monocytes intomonocyte-derived dendritic cells. The results are shown in FIG. 3.

CD14+ human monocyte cells were isolated from human buffy coats usingmagnetic beads as disclosed in Example 1. Cells were plated at 2×10⁶/mlin the presence of 15 kD granulysin (10 nM) and in the presence orabsence of human IL-4 (10 ng/ml) for 5 days. After 5 days, 10 ng/ml ofthe toll-like receptor agonist, lipopolysaccharide (LPS) was added andthe cells were further incubated for an additional 48 hours. The cellswere stained with fluorescent antibodies and analyzed by FACS.

FIG. 3 shows the effect of 15 kD granulysin (10 nM) treatment incombination with IL-4 on the expression of several cell surface markersas measured by florescence. FIGS. 3A, 3B, 3D, 3E and 3F all show asignificant up-regulation of fluorescence in cell surface markers CD86,CD209, CD11b, CD80 and HLA-DR, respectively. As already noted in Example2, the level of fluorescence of CD86 and CD209 was observed to increaseupon incubation with 15 kD granulysin over 48 hours. Here, it isobserved that the level of fluorescence increased further uponincubation of 15 kD granulysin and IL-4 over a period of 5 days, incombination with LPS. Up-regulation of HLA-DR, CD80, CD11b, CD209, andCD86, and down-regulation of CD14 is consistent with the previouslyreported phenotype of mature dendritic cells. (Chapuis et al., Eur. J.Immunol. 27:431-441, 1997; Zhou and Tedder, J. Immunol. 154:3821-3835,1995; Shortman and Liu, Nat. Rev. Immunol. 2:151-161, 2002).

Example 4 Vaccine Adjuvant Protocol

Cell culture studies on the ability of 15 kD granulysin and IL-4 to actas an immunoactivating composition were studied using conventionalmethods known in the art. In this example, 15 kD granulysin and IL-4were co-administered to stimulate differentiation of monocytes.

It is known that monocytes incubated with GM-CSF and IL-4 in vitroresult in the production of dendritic cells (Sallusto and Lanzavecchia,J. Exp. Med. 179:1109-1118, 1994). This combination protocol utilizingGM-CSF and IL-4 is currently the standard vaccine adjuvant protocol asused by the National Institute of Health (NIH), Bethesda, USA, and hasbeen used in clinical studies as a vaccine adjuvant for some time(Belardelli et al., Cancer Res. 34:3827-3830, 2004). Surprisingly, itwas found by the inventors that 15 kD granulysin can be effectivelysubstituted for GM-CSF in the above vaccine adjuvant protocol to providea therapeutically effective and efficient vaccine adjuvant. Overall, theinventors determined that GM-CSF can be readily substituted by 15 kDgranulysin in the vaccination protocol to achieve the same diagnosticand therapeutic effect.

Table 1 shows the results of cell culture studies (CD14+ monocytes)incubated for 4 days with various compositions, stained with fluorescentantibodies and analyzed by FACS analysis. In this example, GM-CSF andIL-4 were used at a concentration of 10 μg/ml. Monocyte cells wereisolated and treated as described in Example 1 and subjected to stainingand analysis by FACS for the following cell surface markers: CD11b,CD14, CD40, CD80, CD83, CD86, CD209, and HLA-DR. It was observed thatthe percentage positive cells was significantly elevated in monocytecells treated with 15 kD granulysin and IL-4, as compared to the controlsample (cell culture medium only) or IL-4 treatment. Importantly, it wasalso observed that the percentage positive cells for cell surfacemarkers CD14, CD83 and CD209 upon incubation with 15 kD granulysin andIL-4 treatment were comparable to the percentage positive cells for thecell surface markers CD14, CD83, and CD209 when incubated with GM-CSFand IL-4.

Table 1 also shows that monocytes incubated with 15 kD granulysin andIL-4 had a slightly elevated percentage of positive cells for CD86 ascompared to the percentage positive cells after incubation with GM-CSFand IL-4. From this in vitro data it is apparent that the expression ofcell surface markers associated with dendritic cells is significantlyelevated when monocytes are incubated with 15 kD granulysin and IL-4. Itis also apparent from the above results that an immune response can bestimulated following incubation of monocytes with 15 kD granulysin andthat this immune response is heightened upon co-incubation with acytokine, such as IL-4, and a toll-like receptor, such aslipopolysaccharide.

TABLE 1 Immunoactivity of 15 kD granulysin and IL-4 Percentage positivecells 15 kD granulysin GM-CSF + Antibody Medium IL-4 (25 nM) + IL-4 IL-4HLA-DR 30 38 64 90 CD40 4 4 24 30 CD86 22 25 65 50 CD209 10 46 84 86CD83 4 10 20 21 CD14 21 14 45 47 CD80 4 5 38 47 CD11b 38 48 48 85

Example 5 Method of Stimulating an Immune Response to an Antigen

In this example, a subject in need of an enhanced immune response to anantigen, for example a tumor antigen is intravenously or subcutaneouslyinfused, following completion of chemotherapy, with a dendritic cellmaturation agent, for example 250 μg/m²/day Leukine™ (a commerciallyavailable form of a yeast-expressed recombinant GM-CSF) as described inthe Leukine™ package insert (Bayer Health Care Pharmaceuticals, 2007).The subject is simultaneously, or within four days of the Leukine™infusion, intravenously administered 10 mg/kg of 15 kD granulysin. Theadministration of 15 kD granulysin and a dendritic cell maturation agentis sufficient to stimulate an immune response against the tumor antigenpresent in the subject.

Example 6 Treatment of Autoimmune Disease

In this example, monocytes are removed from a subject, for example asubject suffering from an autoimmune disease, e.g., Sjogrens Syndrome orautoimmune pancreatitis. The monocytes are manipulated for example, asdescribed in Example 1, to remove non-monocyte cells from the sample.The isolated and purified monocytes are incubated with a therapeuticallyeffective amount of 15 kD granulysin (and optionally, a therapeuticallyeffective amount of a target antigen associated with the autoimmunedisease by which the subject is affected, for example carbonic anhydraseisozyme IV (CA IV)) for a sufficient amount of time to differentiate theisolated monocytes into monocyte-derived dendritic cells (andoptionally, a therapeutically effective amount of a dendritic cellmaturation agent). The monocyte-derived dendritic cells are furtherincubated in vitro with an effective amount of a pharmacological agentcapable of transforming the monocyte-derived dendritic cells totolerogenic dendritic cells, such as TNF-α. The resulting tolerogenicdendritic cells are re-introduced to the subject with the autoimmunedisease in an amount sufficient to treat the symptoms of the autoimmunedisease.

Example 7 Inducing Immunological Tolerance to Organ Transplantation

In this example, monocytes are removed from a subject, for example asubject who is selected to undergo organ transplantation, e.g., kidneytransplantation. The monocytes are manipulated for example, as describedin Example 1, to remove non-monocyte cells from the sample. The isolatedand purified monocytes are incubated with a therapeutically effectiveamount of 15 kD granulysin for a sufficient amount of time todifferentiate the isolated monocytes into monocyte-derived dendriticcells (and optionally, a therapeutically effective amount of a dendriticcell maturation agent). The monocyte-derived dendritic cells are furtherincubated in vitro with a therapeutically effective amount of apharmacological agent capable of transforming the monocyte-deriveddendritic cells into tolerogenic dendritic cells, such as theneuropeptide, vasoactive intestinal peptide (VIP). The resultingtolerogenic dendritic cells are re-introduced to the subject in anamount sufficient to induce immunological tolerance to the transplantedorgan.

Example 8 Treatment of Tumors

In this example, monocytes are removed from a subject, for example asubject diagnosed with, or who is at risk from developing a tumor, e.g.,melanoma. The monocytes are manipulated for example, as described inExample 1, to remove non-monocyte cells from the sample. The isolatedand purified monocytes are incubated with a therapeutically effectiveamount of 15 kD granulysin (and optionally, a therapeutically effectiveamount of a target antigen associated with the tumor the subject has, oris at risk of developing, for example MART-1, MAGE 1 or MAGE 3) for asufficient amount of time to differentiate the isolated monocytes intomonocyte-derived dendritic cells. Optionally, the monocyte-deriveddendritic cells are incubated with one or more dendritic cell maturationagents, for example, IL-4 and/or LPS. The monocyte-derived dendriticcells are re-introduced to the subject in an amount sufficient to treator provide a prophylactic response to the tumor.

Example 9 Treatment of Allergies

In this example, monocytes are removed from a subject, for example asubject diagnosed with an allergy, e.g., dust mite hypersensitivity. Themonocytes are manipulated for example, as described in Example 1, toremove non-monocyte cells from the sample. The isolated and purifiedmonocytes are incubated with a therapeutically effective amount of 15 kDgranulysin (and optionally, a therapeutically effective amount of atarget antigen associated with the allergy, for example Dermatophagoidespteronyssinus (Dp)) for a sufficient amount of time to differentiate theisolated monocytes into monocyte-derived dendritic cells expressing thetarget antigen. Optionally, the monocyte-derived dendritic cellsexpressing the target antigen are incubated with one or more dendriticcell maturation agents, for example, IL-4 and/or LPS. Themonocyte-derived dendritic cells expressing the target antigen arere-introduced to the subject in an amount sufficient to treat or providea prophylactic response to the allergen.

Example 10 GM-CSF Activates Expression of CD1a in Monocytes

Incubation of human CD14+ monocytes with GM-CSF in the presence (orabsence) of IL-4 was found to induce a robust expression of CD1a on thecell surface of the monocytes. However, administration of 15 kDgranulysin was found not to activate expression of CD1a on the surfaceof monocytes. Monocyte cells were prepared and isolated as described inExample 1. Human CD14+ monocytes were incubated with 15 kD granulysin(10 nM) or GM-CSF (10 ng/ml) in the presence or absence of IL-4 (10ng/ml), and incubated for 5 days; cells were subjected to staining forthe cell surface marker CD1a. The cells were analyzed by FACS; theresults of which are presented in FIG. 4A and FIG. 4B.

FIG. 4A discloses the level of cell surface marker expression for CD1aafter culturing with 15 kD granulysin (10 nM), GM-CSF (10 ng/ml), or ascompared to cell culture medium (control). Monocytes treated with 15 kDgranulysin displayed a significant decrease in fluorescence basedexpression of CD1a as compared to GM-CSF treated monocytes. Asignificant increase in fluorescence based expression of cell surfacemarker CD1a was observed when the monocytes were cultured with GM-CSF inthe presence of IL-4 prior to FACS analysis (FIG. 4B). In contrast,monocytes treated with 15 kD granulysin (10 nM) for an equivalent amountof time showed fluorescence profiles similar to the control sample(culture only) or similar to the expression level observed in FIG. 4A.CD1a is an expression marker predominantly associated with the phenotypeof monocytes. Thus, the shift in fluorescence as measured by this cellsurface marker in the presence of GM-CSF is indicative of the cellsretaining monocyte properties and characteristics. In contrast, the cellsurface of human CD14+ monocytes treated with 15 kD granulysin did notappear to be indicative of the cell surface of monocyte cells. Incombination with the data from Examples 2 and 3, the data supports thehypothesis that the human CD14+ monocytes in the presence of 15 kDgranulysin are activated to become monocyte-derived dendritic cells andthereby expression dendritic cell surface markers.

Example 11 15 kD Granulysin Induces Up-Regulation of Cytokine Expressionin Monocytes

Human CD14+ monocytes incubated in the presence of 15 kD were observedto undergo significant up-regulation and/or expression of TNFα, IL-1β,and IL-6 in vitro. Monocyte cells were prepared and isolated asdescribed in Example 1. Human CD14+ monocytes were incubated in cellculture in the presence of 15 kD granulysin (10 nM) or in the presenceof 15 kD granulysin (10 nM) and pertussis toxin (100 ng/ml). The cellswere incubated at 37° C. for 4 hours, after which the cells werecollected, centrifuged and mRNA obtained. The mRNA obtained from thecultured monocytes was converted to cDNA and quantitative PCR wasperformed that allowed for the calculation of fold-increase inexpression of IL-6, IL-1β, or TNFα, relative to a house-keeping gene(β-glucuronidase (GUS)). FIG. 5A-5C are graphs reporting thefold-increase in cytokine expression of IL-6, IL-1β, or TNFα uponadministration of 15 kD granulysin (10 nM) to monocytes in vitro in thepresence or absence of an antigen (pertussis toxin). From the dataprovided in FIG. 5A-5C it was concluded that 15 kD granulysinup-regulates cytokine expression of monocytes in vitro, even in theabsence of a target antigen.

Example 12 Activation and Stimulation of Allogeneic T Cells

CD14+ human monocyte cells were isolated from human buffy coats usingmagnetic beads as disclosed in Example 1. Cells were plated at 2×10⁶/mlin the presence of 15 kD granulysin (10 nM) or in the presence of GM-CSF(10 ng/ml) and incubated for 4 days. After 4 days, 100 ng/ml of thetoll-like receptor agonist, lipopolysaccharide (LPS) was added to thecells to induce dendritic cell maturation and the cells were furtherincubated for an additional two days. On day 6, the cells were harvestedand used to stimulate allogeneic T cells. After five additional days,cellular proliferation was measured and reported as fold-stimulationabove T-cells alone, the results of which are presented in FIG. 6. Thefour upper rows of FIG. 6 represent monocytes activated upon incubationwith GM-CSF (10 ng/ml) or GM-CSF and IL-4 (10 ng/ml). The four lowerrows of FIG. 6 demonstrate fold-stimulation of allospecific T cells uponincubation of human CD14+ monocytes with 15 kD granulysin (10 nM) or 15kD granulysin and IL-4 (10 ng/ml). The data from FIG. 6 demonstratesthat 15 kD granulysin (10 nM) was sufficient to induce a robuststimulation of allogeneic T cells in vitro. Furthermore, thefold-stimulation of allogeneic T cells produced as a result ofincubation with 15 kD granulysin (alone), as compared to the level offold-stimulation induced by GM-CSF (alone), was significantly higher. Incontrast, the level of fold-stimulation of allogeneic T cells uponincubation with 15 kD granulysin and IL-4 was substantially reduced ascompared to the fold-stimulation effects of 15 kD granulysin alone.Overall, the effect of LPS in these test experiments resulted in anobserved decrease of fold-stimulation in allogeneic T cells as comparedto the corresponding LPS-free cultures.

Example 13 15 kD Granulysin Activates Monocytes

Many characteristics of 9 kD granulysin are known in the art, includingits chemoattractant property. To determine if 15 kD granulysin possesseschemotactic potential, particularly with respect to monocytes, thefollowing experiment was performed. Monocytes are cells of the immunesystem and in response to inflammatory move to the site of infectionwhere they can divide into dendritic cells or macrophages. The presenceof monocytes at the site of infection might be a direct consequence ofthe properties of 15 kD granulysin in response to inflammation or isperhaps a mechanism by which a shift in the dual-production pathway ofmacrophages and dendritic cells is converted into a predominantly singlepathway, e.g., the production of dendritic cells.

CD14+ human monocyte cells were isolated from human buffy coats usingmagnetic beads as disclosed in Example 1. Human CD14+ monocytes werecultured for 6 hours at 37° C. in cell culture medium alone, or in cellculture medium supplemented with 15 kD granulysin (10 nM). After 6hours, cells were visualized using 60× magnification. Monocytesincubated solely in the presence of cell culture medium were dispersedrandomly across the visual field and were not observed to aggregate. Incontrast, the monocytes incubated in cell culture medium supplementedwith 15 kD granulysin were observed to form significant cell clusters.

Example 14 Pharmaceutical Formulations of 15 kD Granulysin

In one embodiment, a therapeutically effective amount of 15 kDgranulysin is formulated for administration to the skin. Formulationssuitable for topical administration can include dusting powders,ointments, creams, gels, sprays, or transdermal patches for theadministration of 15 kD granulysin to cells, such as skin cells. Topicalformulations may be administered to blisters or lesions present on theskin, such as leprosy lesions and blisters. In one example, the 15 kDgranulysin is administered to the skin for the treatment of melanoma.Alternatively, the 15 kD granulysin is co-administered with a vaccine,for example, an Alzheimer's vaccine, to a host in the form of atransdermal patch. While not wishing to be bound by the following, it isbelieved that the vaccine and 15 kD granulysin work in combination toactivate and/or up-regulate the immune system of the host to recognize atarget antigen, such as beta amyloid protein (Aβ) which abnormallyaccumulates in the brains of Alzheimer's patients. Activation of thesubject's immune response following recognition of the target antigenallows the immune system to recognize and inactivate the antigen,thereby reducing or treating the pathology.

The pharmaceutical formulations may optionally include an inorganicpigment, organic pigment, inorganic powder, organic powder, hydrocarbon,silicone, ester, triglyceride, lanolin, wax, cere, animal or vegetableoil, surfactant, polyhydric alcohol, sugar, vitamin, amino acid,antioxidant, free radical scavenger, ultraviolet light blocker,sunscreen agents, preservative, fragrance, thickener, or a combinationthereof.

In one example, 15 kD granulysin can be used in cosmetic formulations(e.g., skincare cream, sunscreen, decorative make-up products, and otherdermatological compositions) in various pharmaceutical dosage forms, andin the form of oil-in-water or water-in-oil emulsions, solutions, gels,or vesicular dispersions. The cosmetic formulations may take the form ofa cream which can be applied either to the face or to the scalp andhair, as well as to the human body, in particular those portions of thebody that are chronically exposed to sun or an environmental carcinogen.The cosmetic formulation can also serve as a base for a lip-gloss orlipstick.

In some cosmetic formulations, additives can be included such as, forexample, preservatives, bactericides, perfumes, antifoams, dyes,pigments which have a coloring action, surfactants, thickeners,suspending agents, fillers, moisturizers, humectants, fats, oils, waxesor other customary constituents of a cosmetic formulation, such asalcohols, polyols, polymers, foam stabilizers, electrolytes, organicsolvents, or silicone derivatives.

Cosmetic formulations typically include a lipid phase and often anaqueous phase. The lipid phase can be chosen from the following group ofsubstances: mineral oils, mineral waxes, such as triglycerides of capricor of caprylic acid, castor oil; fats, waxes and other natural andsynthetic fatty substances, esters of fatty acids with alcohols of low Cnumber, for example with isopropanol, propylene glycol or glycerol, oresters of fatty alcohols with alkanoic acids of low C number or withfatty acids; alkyl benzoates; silicone oils, such asdimethylpolysiloxanes, diethylpolysiloxanes, diphenylpolysiloxanes andmixed forms thereof.

If appropriate, the aqueous phase of the formulations according to thepresent disclosure include alcohols, diols or polyols of low C numberand ethers thereof, such as ethanol, isopropanol, propylene glycol,glycerol, ethylene glycol, ethylene glycol monoethyl or monobutyl ether,propylene glycol monomethyl, monoethyl or monobutyl ether, diethyleneglycol monomethyl or monoethyl ether and analogous products, furthermorealcohols of low C number, for example ethanol, isopropanol,1,2-propanediol and glycerol, and, in particular, one or morethickeners, such as silicon dioxide, aluminum silicates, polysaccharidesand derivatives thereof, for example hyaluronic acid, xanthan gum andhydroxypropylmethylcellulose, or poly-acrylates.

An exemplary 15 kD granulysin cosmetic formulation is as an additive toa sunscreen composition as a lotion, spray or gel, for administration tothe skin. A sunscreen can additionally include at least one further UVAfilter and/or at least one further UVB filter and/or at least oneinorganic pigment, such as an inorganic micropigment. The UVB filterscan be oil-soluble or water-soluble. Oil-soluble UVB filter substancescan include, for example: 3-benzylidenecamphor derivatives, such as3-(4-methylbenzylidene)camphor and 3-benzylidenecamphor; 4-aminobenzoicacid derivatives, such as 2-ethylhexyl 4-(dimethylamino)benzoate andamyl 4-(dimethylamino)benzoate; esters of cinnamic acid, such as2-ethylhexyl 4-methoxycinnamate and isopentyl 4-methoxycinnamate;derivatives of benzophenone, such as 2-hydroxy-4-methoxybenzophenone,2-hydroxy-4-methoxy-4′-methylbenzophenone and2,2′-dihydroxy-4-methoxybenzophenone; esters of benzalmalonic acid, suchas di(2-ethylhexyl)4-methoxybenzalmalonate. Water-soluble UVB filtersubstances can include the following: salts of2-phenylbenzimidazole-5-sulphonic acid, such as its sodium, potassium orits triethanolammonium salt, and the sulphonic acid itself; sulphonicacid derivatives of benzophenones, such as2-hydroxy-4-methoxybenzophenone-5-sulphonic acid and salts thereof;sulphonic acid derivatives of 3-benzylidenecamphor, such as, forexample, 4-(2-oxo-3-bornylidenemethyl)benzenesulphonic acid,2-methyl-5-(2-oxo-3-bornylidenemethyl)benzenesulphonic acid and saltsthereof. The list of further UVB filters mentioned which can be used incombination with 15 kD granulysin according to the disclosure is notintended to be limiting.

For treatment of the skin, a therapeutically effective amount of 15 kDgranulysin can be locally administered to an affected area of the skin,such as in the form of an ointment. In one embodiment, the ointment isan entirely homogenous semi-solid external agent with firmnessappropriate for easy application to the skin. The ointment can includefats, fatty oils, lanoline, Vaseline, paraffin, wax, hard ointments,resins, plastics, glycols, higher alcohols, glycerol, water, emulsifieror a suspending agent. Using these ingredients as a base, a decoycompound can be evenly mixed. Depending on the base, the mixture can bein the form of an oleaginous ointment, an emulsified ointment, or awater-soluble ointment. Oleaginous ointments use bases such as plant andanimal oils and fats, wax, Vaseline and liquid paraffin. Emulsifiedointments are comprised of an oleaginous substance and water, emulsifiedwith an emulsifier. They can take either an oil-in-water form (O/W) or awater-in-oil-form (W/O). The oil-in-water form (O/W) can be ahydrophilic ointment. The water-in-oil form (W/O) initially lacks anaqueous phase and can include hydrophilic Vaseline and purifiedlanoline, or it can contain a water-absorption ointment (including anaqueous phase) and hydrated lanoline. A water-soluble ointment cancontain a completely water-soluble Macrogol base as its main ingredient.Alternatively, the 15 kD granulysin can be administered as a transdermalpatch that can deliver sustained therapeutic levels of 15 kD granulysinthrough the skin in a convenient, painless manner, for example, usingthe PassPort Transdermal System™ developed by Altea Therapeutics(Atlanta, Ga.). In a further embodiment, the 15 kD granulysintransdermal patch may include one or more therapeutic compounds, forexample, a vaccine, a drug (such as a chemotherapeutic drug), ananti-inflammatory compound, or other therapeutic agent.

In other embodiments, the 15 kD granulysin can be formulated in anaqueous solution, preferably in a physiologically compatible buffer. Fortransmucosal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art. For oral administration, 15 kD granulysin can becombined with carriers suitable for inclusion into tablets, pills,dragees, capsules, caplets, liquids, gels, syrups, slurries, suspensionsand the like. The 15 kD granulysin can also be formulated for use ininhalation therapy. For administration by inhalation, 15 kD granulysinis conveniently delivered in the form of an aerosol spray presentationfrom pressurized packs or a nebulizer, with the use of a suitablepropellant. The 15 kD granulysin can be formulated for parenteraladministration by injection, e.g., by bolus injection or continuousinfusion. Similarly, a composition including 15 kD granulysin can beformulated for intratracheal or for inhalation administration. Suchcompositions can take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and can contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Other pharmacologicalexcipients are known in the art.

In one embodiment, 15 kD granulysin is applied with, or as part of acomposition including a pharmaceutically acceptable carrier, diluent orexcipient (including combinations thereof). The carrier, diluent orexcipient must be “acceptable” in the sense of being compatible with thecomposition of the invention and not deleterious to the recipientthereof. Typically, the carriers will be water or saline which will besterile and pyrogen free. Acceptable carriers or diluents fortherapeutic use are well known in the pharmaceutical art, and aredescribed, for example, in Remington: The Science and Practice ofPharmacy, The University of the Sciences in Philadelphia, Editor,Lippincott, Williams, & Wilkins, Philadelphia, Pa., 21^(st) Edition(2005). The choice of pharmaceutical carrier, excipient or diluent canbe selected with regard to the intended route of administration andstandard pharmaceutical practice. The pharmaceutical composition maycomprise as, or in addition to, the carrier, excipient or diluent anysuitable binder, lubricant, suspending agent, coating agent,solubilizing agent or combinations thereof.

Pharmaceutically acceptable carriers include a petroleum jelly, such asVASELINE®, wherein the petroleum jelly contains 5% stearyl alcohol, orpetroleum jelly alone, or petroleum jelly containing liquid paraffin.Such carriers enable pharmaceutical compositions to be prescribed informs appropriate for consumption, such as tablets, pills, sugar-coatedagents, capsules, liquid preparations, caplets, gels, ointments, syrups,slurries, and suspensions. When locally administered to cells in anaffected area or tissue of interest, the 15 kD granulysin compositioncan be administered in a formulation that contains a synthetic ornatural hydrophilic polymer as the carrier. Examples of such polymersinclude hydroxypropyl cellulose and polyethylene glycol. The compositioncan be mixed with a hydrophilic polymer in an appropriate solvent. Thesolvent is then removed by methods such as air-drying, and the remainderis then shaped into a desired form (for example, a sheet) and applied tothe target site. Formulations containing such hydrophilic polymers keepwell as they have a low water-content. At the time of use, theformulation absorbs water, becoming a gel that also stores well. In thecase of sheets, the firmness can be adjusted by mixing a polyhydricalcohol with a hydrophilic polymer similar to those above, such ascellulose, starch and its derivatives, or synthetic polymeric compounds.Accordingly, a therapeutically effective amount of 15 kD granulysin canbe incorporated into bandages, plasters, transdermal patches or otherwound dressings.

Example 15 Modes of Administration of 15 kD Granulysin

According to the disclosed methods, compositions of the presentinvention can be administered by, but not limited to, intramuscular(i.m.), intravenously (i.v.), subcutaneous (s.c.), or intrapulmonaryroutes. Transdermal delivery includes, but is not limited to intradermal(e.g., into the dermis or epidermis), transdermal (e.g., percutaneous)and transmucosal administration (e.g., into or through skin or mucosaltissue). Intracavity administration includes oral, nasal, peritoneal,rectal, vaginal or intestinal cavities as well as, intrathecal,intraventricular, intraarterial and sub arachnoid administration.

Any mode of administration is contemplated so long as the mode resultsin the activation of an immune response to the target antigen, in thedesired tissue, in an amount sufficient to generate a therapeutically orprophylactically effective immune response against the target antigen.

Determining an effective amount of the composition depends on a numberof factors including, for example, the antigen being expressed oradministered directly, the age and weight of the subject, the precisecondition requiring treatment, the severity of the condition, and theroute of administration. Based on the above factors, determining theprecise amount, number of doses, and timing of doses are within theordinary skill in the art and will be readily determined by theattending physician or veterinarian.

The 15 kD granulysin can be formulated for administration by inhalation,such as, but not limited to, formulations for the treatment of lung oresophageal cancer. Inhalational preparations include aerosols,particulates, and the like. In general, the goal for particle size forinhalation is about 1 μm or less in order that the composition reachesthe alveolar region of the lung for absorption. However, the particlesize can be modified to adjust the region of disposition in the lung.Thus, larger particles can be utilized (such as about 1 to about 5 μm indiameter) to achieve deposition in the respiratory bronchioles and airspaces. In addition, oral formulations may be liquid (e.g., syrups,solutions, or suspensions), or solid (e.g., powders, pills, tablets,caplets, or capsules).

For administration by inhalation, the compositions can be convenientlydelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebulizer, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit can be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesfor use in an inhaler or insufflator can be formulated containing apowder mix of the composition and a suitable powder base such as lactoseor starch.

When 15 kD granulysin is provided as a parenteral composition, e.g. forinjection or infusion, it is generally suspended in an aqueous carrier,for example, as an isotonic buffer solution at a pH of about 3.0 toabout 8.0, preferably at a pH of about 3.5 to about 7.4, more preferablyat about 3.5 to about 6.0, or most preferably between about 3.5 andabout 5.0. Useful buffers include sodium citrate-citric acid and sodiumphosphate-phosphoric acid, and sodium acetate-acetic acid buffers. Aform of repository or “depot” slow release preparations may also be usedso that therapeutically effective amounts of 15 kD granulysin aredelivered into the blood vessels over many hours or days followingtransdermal administration or delivery.

The 15 kD granulysin composition can be administered as asustained-release system, for example of sustained-release compositionsincluding suitable polymeric materials (such as, for example,semi-permeable polymer matrices in the form of shaped articles, e.g.,films, or mirocapsules), suitable hydrophobic materials (such as, forexample, an emulsion in an acceptable oil) or ion exchange resins, andsparingly soluble derivatives (such as, for example, a sparingly solublesalt). Sustained-release compositions as described herein may beadministered orally, rectally, parenterally, intravaginally,intraperitoneally, topically (as by powders, ointments, gels, drops ortransdermal patch), bucally, or as an oral or nasal spray.

Preparations for administration can be suitably formulated to givecontrolled release of 15 kD granulysin over an extended period of time.For example, the pharmaceutical composition may be in the form ofparticles including a biodegradable polymer and/or a polysaccharidejellifying and/or bioadhesive polymer, an amphiphilic polymer, an agentmodifying the interface properties of the particles and apharmacologically active substance. These compositions exhibit certainbiocompatibility features which allow a controlled release of an activeingredient, as described in U.S. Pat. No. 5,700,486.

For oral administration, the pharmaceutical composition including 15 kDgranulysin can take the form of, for example, tablets or capsulesprepared by conventional means with pharmaceutically acceptableexcipients such as binding agents (for example, pregelatinized maizestarch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers(for example, lactose, microcrystalline cellulose or calcium hydrogenphosphate); lubricants (for example, magnesium stearate, talc orsilica); disintegrants (for example, potato starch or sodium starchglycolate); or wetting agents (for example, sodium lauryl sulfate). Thetablets can be coated by methods well known in the art. In someinstances, the disclosed compositions may be microencapsulated (e.g.,poly(DL lactide-coglycolide) to reduce or prevent significantdegradation of the composition prior to reaching the small intestine.Indeed, oral immunization with antigen incorporated in microparticleshas been demonstrated to induce systemic and secretory antibodyresponses (Eldridge et al., Curr. Top. Microbiol. Immunol. 146:59-66,1989; Challacombe et al., Immunol. 76:164-168, 1992). Actual methods ofpreparing such dosage forms are known, or will be apparent, to those ofordinary skill in the art.

The pharmaceutically acceptable carrier and excipient useful in thisinvention are conventional. For instance, parenteral formulationsusually comprise injectable fluids that are pharmaceutically andphysiologically acceptable such as water, physiological saline, otherbalanced salt solutions, aqueous dextrose, glycerol or the like. Ifdesired, the pharmaceutical composition may also contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, pH buffering agents, and the like, for examplesodium acetate or sorbitan monolaurate. Actual methods of preparing suchdosage forms are known, or will be apparent, to those skilled in theart.

Generally, the formulations are prepared by contacting 15 kD granulysinuniformly and intimately with liquid carriers or finely divided solidcarriers or both. Then, if necessary, the product is shaped into thedesired formulation. Optionally, the carrier is a parenteral carrier,and in some embodiments it is a solution that is isotonic with the bloodof the recipient. Examples of such carrier vehicles include water,saline, Ringer's solution, and dextrose solution. Non-aqueous vehiclessuch as fixed oils and ethyl oleate are also useful herein, as well asliposomes. Compositions of the present invention may include variousexcipients, carriers and/or delivery vehicles as are disclosed, e.g., inU.S. Patent Application No. 2002/0019358, published Feb. 14, 2002, whichis incorporated herein by reference to the extent it discloses suchcompositions.

The 15 kD granulysin pharmaceutical composition can be formulated inunit dosage forms suitable for individual administration of precisedosages. The amount administered will be dependent on the subject beingtreated, the severity of the condition or disorder, and the manner ofadministration, and is best left to the judgment of the prescribingclinician. Within these bounds, the formulation to be administered willcontain a quantity of 15 kD granulysin in an amount effective to achievethe desired effect in the subject being treated. In some incidences,multiple treatments are envisioned, such as over a defined interval oftime, for example as daily, bi-weekly, weekly, bi-monthly or monthlyadministration, such that chronic administration is achieved. Asdisclosed herein, a therapeutically effective amount of 15 kD granulysincan be used to inhibit the formation of a tumor, treat a tumor, inhibitconversion of a benign tumor to a malignant tumor, decrease the risk ofdeveloping a tumor, or inhibit metastasis. Administration of the 15 kDgranulysin composition may begin whenever the suppression or inhibitionof disease is desired, for example, at a certain age of a subject, orprior to an environmental exposure.

Example 16 Comparison of 15 kD Granulysin and GM-CSF In Vitro

Expression and purification of 15 kD granulysin: A cDNA clone of 15 kDgranulysin was generated from human peripheral blood and cloned intopet28A E. coli expression vector. A baculovirus GP67 secretion leaderwas engineered at the 5′ end of the granulysin gene by adapter PCR. Theverified clone was subcloned by Gateway LR recombination (Invitrogen,Carlsbad, Calif.) into pDest-670 for insect cell expression. Theexpression clone was then transformed in to E. coli DH10Bac(Invitrogen), and plated on LB medium containing kanamycin, gentamycin,tetracycline, X-gal, and IPTG as per manufacturer's protocols. Thebacmid DNA was verified by PCR amplification across the bacmid junctionsand transfected into SF-9 insect cells to create the recombinantbaculovirus. Large scale expression was done using Hi5 insect cellsgrown in 3 L Erlenmeyer flasks. Cells were infected at a multiplicity ofinfection of three, maintained at 27° C. for 4 hours, then shifted to21° C. and allowed to grow for 48 hours. Cells were spun out and thesupernatant containing the secreted 15 kD granulysin was filtered usinga 0.45 μM filter and stored at −20° C. This material was applied to a 5ml HiTrap™ Heparin HP (GE Health Care, Uppsala, Sweden). Fractionscontaining the 15 kD granulysin were pooled, buffer exchanged, and runon a 1 ml Resource S column (GE Health Care). The purified protein wasconcentrated and stored at −80° C. Finn et al., Protein Expr. Purif.doi:10.1016/j.pep.2010.07.009, 2010.

Activation of monocytes and flow cytometry: Elutriated human monocyteswere cultured at 2×10⁶ cells/ml in 24 well plates in RPMI-1640supplemented with 10% heat-inactivated FBS (Hyclone, Ogden, Utah), 2 mML-glutamine, and 100 U/ml penicillin-streptomycin (complete medium). 15kD granulysin (10 nM), GM-CSF (10 ng/ml), and IL-4 (10 ng/ml) were addedas described. Cells were stained with the following antibodies from BDBioscience (San Diego, Calif.): CD86 (clone FUN-1) and CD83 (HB15e) asfluorescein isothiocyanate (FITC) conjugates; CD14 (M5E2), CD80(L307.4), and CD209 (DCN46) as phycoerythrin (PE) conjugates; CD11c(Bly6), CD11b (ICRF44), CD40 (5C3), and CD1a (HI149) as APC conjugates;and from eBiosciences (San Diego, Calif.) CD1c (L161) FITC conjugate andHLA-DR (L243) as PE conjugates. In some cases, cells were then fixed andpermeabilized using BD Cytofix/Cytoperm™ and then stained withantibodies specific for IL-6 (MQ2-6A3) PE conjugate, TNF (MAb11) AF488conjugate, interferon-γ (B27) PeCy7 conjugate (all from BD Biosciences)or IL1β (CRM56) FITC conjugate from eBiosciences. Flow cytometry datawas analyzed with FlowJo analysis software (Tree Star, Ashland, Oreg.).

Using elutriated CD14+ monocytes, the effects of 15 kD granulysin andGM-CSF, a well-characterized activator of monocytes, were compared(FIGS. 8A-8C). Within 6 hours, monocytes cultured with 10 nM 15 kDgranulysin, but not with 10 ng/ml GM-CSF, formed aggregates (FIG. 8A).Both 15 kD granulysin and GM-CSF caused an increase in cell size andupregulation of adhesion molecules including CD11b, CD11c, and CD54, aswell as molecules associated with differentiation to immature dendriticcells, including CD40, CD80, CD86 and HLA-DR (FIG. 8B). 15 kDgranulysin, but not GM-CSF, promoted increased expression of CD83 whileGM-CSF, but not 15 kD granulysin, caused increased expression of CD1aand CD1c. 15 kD granulysin, but not GM-CSF, also caused a rapid increasein expression of IL-1β, IL-6, and TNFα in monocytes (FIG. 8C).

Of note, 15 kD granulysin also activated immature dendritic cells.Monocytes cultured with GM-CSF plus IL-4 for 4 days and then treatedwith 15 kD granulysin for another 24 hours expressed even higher levelsof CD40, CD80, CD83, CD86, and HLA-DR, characteristic of maturedendritic cells (FIG. 9A). The stimulatory capabilities of dendriticcells generated with 15 kD granulysin were evaluated in two ways: (1)proliferation was increased 60-fold when T cells were incubated withallogeneic dendritic cells activated by 15 kD granulysin and (2)coculture of allogeneic T cells with 15 kD activated mature dendriticcells resulted in T cells producing TNFα and IFNγ, but not IL-6 or IL-4,suggesting that 15 kD granulysin induced dendritic cells to a state thatfavored Th1 over Th2 or Th17 T cell differentiation (FIG. 9B).

Example 17 Effect of 15 kD Granulysin on Monocyte Gene Expression

Because the dendritic cells generated from monocytes in vitro using 15kD granulysin differ in some ways from those dendritic cells generatedwith GM-CSF (Example 16, above), microarrays were used to compare theeffects of 15 kD granulysin and GM-CSF on gene expression in humanmonocytes. Total RNA was extracted using Trizol (Invitrogen, Carlsbad,Calif.) from elutriated monocytes cultured as described in Example 16.RNA integrity was assessed using an Agilent 2100 Bioanalyser (AgilentTechnologies, Waldbronn, Germany). Test samples (500 ng) and UniversalReference tRNA (500 ng, Invitrogen) were processed using an Agilent kit,labeled with Cy5 and Cy3, respectively, and co-hybridized according tothe manufacturer's instructions on Agilent Chips (Agilent Technologies,Whole Human genome, 4×44 k). Microarray image analysis was performedusing Agilent Feature Extraction Software 9.5.1.1. The resultingnormalized data were uploaded on mAdb Gateway (madb.nci.nih.gov) andfurther analyzed using BRB Array Tools(linus.nci.nih.gov/BRB-ArrayTools.html), which was developed at theNational Cancer Institute (NCI), Biometric Research Branch, Division ofCancer Treatment and Diagnosis (Simon et al., Cancer Inform. 3, Feb. 4,2007). The data set was filtered according to a standard procedure toexclude spots below a minimum intensity that was set to an arbitraryintensity parameter of 20 in both fluorescence channels. Of these,33,757 genes passed the filter and were used for further analysis.Hierarchical cluster analysis and TreeView software were used forvisualization of the data (http://rana.lbl.gov; Eisen et al., Proc.Natl. Acad. Sci. USA 95:14863, 1998). Class comparison analysis wasconducted at a p-value <0.001, random variance model and univariatepermutation tests were included to strengthen the analysis.

Global gene expression was assessed pre-treatment (time 0) and after 4,12, 24 and 48 hours of treatment with either 10 ng/ml GM-CSF or 10 nM 15kD granulysin in monocytes obtained from 3 subjects. 6103 genes werestatistically differentially expressed and showed similar patterns ofexpression by monocytes treated with either GM-CSF or 15 kD granulysin(p-value <0.001) when compared to their expression levels inpre-treatment monocytes. However, a direct comparison between GM-CSF andgranulysin treated monocytes at each time point showed a total of 3690genes differentially expressed (p-value <0.001) between the twotreatments. Of these, the expression of 1815 genes was greater in 15 kDgranulysin treated monocytes while expression of 1875 genes was greaterin monocytes treated with GM-CSF.

Chemokine/cytokine and costimulatory/adhesion genes induced by 15 kDgranulysin were selected for further analysis. Genes that increased inexpression at least 5-fold in the microarray analysis were selected andtheir mRNA levels were determined by real time qPCR. Elutriatedmonocytes were cultured with 10 nM 15 kD granulysin or 10 ng/ml GM-CSFas in Example 16 and cells were harvested at 4, 12 and 24 hours andfrozen. RNA was prepared using an RNeasy® MiniKit and Qiashreddercolumns (Qiagen, Valencia, Calif.). cDNA was generated using theiScript™ cDNA Synthesis kit (BioRad, Hercules, Calif.) using themanufacturer's suggested protocol. rtPCR reactions were set up in 384well plates (Applied Biosystems, Foster City, Calif.) in a finalreaction volume of 10 The reaction contained the Power SyBR® Green PCRMaster Mix (Applied Biosystems). PCR was conducted using a 7900HT FastReal-Time PCR System (Applied Biosystems) and data were analyzed usingSDS 2.3 software package (Applied Biosystems). GUS was used as thecontrol gene for each time point.

As shown in Table 2, mRNA for all these genes was increased over levelsin cells cultured in medium alone. In contrast, at the four-hour timepoint, only CD274 and CD80 were slightly upregulated in monocytescultured with GM-CSF. At 4, 12 and 24 hours, the majority of these geneswere expressed at much higher levels in cells treated with 15 kDgranulysin than in those treated with GM-CSF. Protein expression wasalso confirmed for a subset of these genes. At 24 hours, monocytescultured with 15 kD granulysin expressed abundant levels of IL-1β, IL-6and TNFα while cells cultured with GM-CSF did not express thesecytokines (Table 2). These data indicate that 15 kD granulysin affectsmonocytes differently from GM-CSF, suggesting that 15 kD granulysin maybe a useful alternative for production of antigen presenting cells foradoptive cell based therapies.

TABLE 2 Immune-related gene expression in monocytes activated by 15 kDgranulysin or GMCSF 15 kD Granulysin GM-CSF 4 hour 12 hour 24 hour 4hour 12 hour 24 hour IL-6 2802 3308 940 1 3 2 CCL20 243 375 1056 0 1 1TNFAIP6 62 100 31 2 1 0 CXCL1 66 131 16 1 2 0 ITGB8 124 220 36 2 1 0TNFAIP8 22 5 5 3 1 1 TNF 35 31 6 1 3 3 CXCL2 19 81 51 1 3 1 TRAF1 20 215 1 1 0 CXCL3 16 83 41 1 1 1 IL7R 8 23 8 2 2 4 ADAMDECH1 4 22 8 1 1 0TNFRSF4 11 24 2 1 3 1 CD274 319 102 12 11 7 2 CD80 35 42 21 6 17 17 SPP17 23 16 2 12 26 IL1B 70 206 248 1 6 8 CCL23 350 1013 311 2 7 143 MMP14158 194 55 1 1 5 CCL2 731 48 8 1 2 43 CCL7 79 19 8 1 1 18

Example 18 Effect of 15 kD Granulysin Expression on Tumors In Vivo

To further investigate a role for 15 kD granulysin in clinicalsituations, a mouse model was utilized. Mice do not have a granulysinhomologue, however, mice transgenic for human granulysin have beengenerated (Huang et al., J. Immunol. 178:77-84, 2007). It was previouslyshown that allospecific T cell lines generated from granulysintransgenic animals showed enhanced killing of target cells. In vivoeffects of granulysin have been evaluated using the syngeneic T lymphomatumor C6VL. Granulysin transgenic mice survived significantly longerthan nontransgenic littermates in response to a lethal tumor challenge(Huang et al., J. Immunol. 178:77-84, 2007). These findings demonstratedfor the first time an in vivo effect of granulysin. To build upon theseresults in another model with a different strain, C57BL/6 GNLY^(+/−)animals were crossed onto Balb/c mice.

WT and GNLY^(+/−) mice were injected in the right flank with 1.5×10⁶CT26 tumor cells. After 12-14 days, tumors were removed and weighed.Flow cytometry was performed using the following antibodies from BDBiosciences: FITC-conjugated CD3 (145-2C11), CD4 (L3T4), CD8 (53-6.7);PE-conjugated CD40 (3/23) and CD86 (GL1); APC-conjugated interferon-γ(XMG1.2) and TNF (MP6-XT22). Tumor infiltrating lymphocytes (TIL) andlymphocytes from the draining inguinal and popliteal lymph nodes wereprepared and analyzed for expression of CD40 and CD86. TIL were alsosubjected to intracellular staining for TNFα and IFNγ following in vitrostimulation with PMA and ionomycin in the presence of GolgiStop™ (BDBiosciences).

After >10 backcrosses, the animals were challenged with the syngeneicCT26 colon carcinoma (Wu et al., Med. Oncol. 27:736-742, 2010). Tumorsfrom both wild type and transgenic mice were removed at various timesand tumor-infiltrating lymphocytes (TIL) were prepared. In addition, thedraining lymph nodes were removed for analysis. TIL from GNLY^(+/−) miceexpressed granulysin while those from wild type mice did not. UsingWestern blot of TIL, both 9 kD and 15 kD granulysin were observed, withthe 9 kD isoform predominating. Cytokine production and activationmarker expression were examined by flow cytometry. GNLY^(+/−) mice hadsignificantly smaller tumor nodules than wild type mice (FIG. 10A), andthis correlated with higher levels of IFNγ and TNF production inlymphocytes isolated from the draining nodes (FIG. 10B). Furthermore,antigen-presenting cells from both draining lymph nodes and tumornodules from the GNLY^(+/−) mice expressed higher levels of CD40 andCD86 (FIG. 10C), indicating that the capacity of T cells to producegranulysin correlates with enhanced costimulatory/coactivatingproperties of antigen presenting cells in vivo.

Example 19 Effect of 15 kD Granulysin in Primates

The effect of 15 kD granulysin on monocyte differentiation in vitro canbe determined in non-human primates. Immune responses can also bedetermined in vivo in non-human primates, utilizing ex vivo monocytecells differentiated to dendritic cells in the presence of 15 kDgranulysin, co-administered with an antigen. Exemplary methods aredescribed, however, one skilled in the art will appreciate that methodsthat deviate from these specific methods can also be used tosuccessfully assess the effect of 15 kD granulysin in a primate model.

Peripheral blood mononuclear cells (PBMC) or purified monocytes arecollected from rhesus macaques and are cultured with 15 kD granulysin(such as about 1 nM to about 1 μM) or GM-CSF (such as about 10 ng/ml)for 1-5 days. Cells are then stained for cell surface antigens specificto various cell types, such as T cells, B cells, monocytes, NK cells,and dendritic cells. The ability of 15 kD granulysin to promotedifferentiation of monocytes to immature dendritic cells is assessed bydetecting cell surface markers of immature dendritic cells followingtreatment with 15 kD granulysin.

Monocytes purified from rhesus macaques are cultured with 10 ng/mlGM-CSF and IL-4 (10 ng/ml) for 2-3 days and then 15 kD granulysin isadded for about 24 hours. Cell surface markers of mature dendritic cellsare assessed (such as CD40, CD80, CD83, and CD86).

Cytokine expression is also assessed in the 15 kDgranulysin-differentiated cells. Dendritic cells activated with 15 kDgranulysin or GM-CSF are cocultured with allogeneic T cells andexpression of TNFα, IFNγ, IL-6, and 11-4 are measured to assess T celldifferentiation.

Dendritic cells activated ex vivo with 15 kD granulysin as describedabove are infused into rhesus macaques with antigen to monitor theeffect on in vivo responses.

In view of the many possible embodiments to which the principles of thedisclosure may be applied, it should be recognized that the illustratedembodiments are only examples and should not be taken as limiting thescope of the invention. Rather, the scope of the invention is defined bythe following claims. We therefore claim as our invention all that comeswithin the scope and spirit of these claims.

What is claimed is:
 1. A method of inhibiting development of a tumor ina subject, comprising: selecting a subject who is at risk for developinga tumor, and administering to the subject at risk of developing thetumor a therapeutically effective amount of a tumor antigen and 15 kDgranulysin for a sufficient amount of time to inhibit development of thetumor in the subject.
 2. The method of claim 1, further comprisingadministering to the subject a therapeutically effective amount of achemotherapeutic or anti-inflammatory agent.
 3. The method of claim 1,wherein the tumor is a benign or malignant tumor.
 4. The method of claim3, wherein the tumor is a brain, a gastrointestinal, an esophageal, astomach, a lung, a liver, a kidney, a skin or a colon tumor.